Modulators of amyloid aggregation

ABSTRACT

Compounds that modulate the aggregation of amyloidogenic proteins or peptides are disclosed. The modulators of the invention can promote amyloid aggregation or, more preferably, can inhibit natural amyloid aggregation. In a preferred embodiment, the compounds modulate the aggregation of natural β amyloid peptides (β-AP). In a preferred embodiment, the β amyloid modulator compounds of the invention are comprised of an Aβ aggregation core domain and a modifying group coupled thereto such that the compound alters the aggregation or inhibits the neurotoxicity of natural β amyloid peptides when contacted with the peptides. Furthermore, the modulators are capable of altering natural β-AP aggregation when the natural β-APs are in a molar excess amount relative to the modulators. Pharmaceutical compositions comprising the compounds of the invention, and diagnostic and treatment methods for amyloidogenic diseases using the compounds of the invention, are also disclosed.

RELATED APPLICATIONS

This application is a continuation of patent application Ser. No.09/972,475, filed on Oct. 4, 2001, which is a continuation of patentapplication Ser. No. 08/617,267, filed on Mar. 14, 1996, which is acontinuation-in-part of U.S. patent application Ser. No. 08/404,831,filed Mar. 14, 1995, and U.S. patent application Ser. No. 08/475,579,filed Jun. 7, 1995 and U.S. patent application Ser. No. 08/548,988,filed Oct. 27, 1995, the entire contents of each of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD), first described by the Bavarian psychiatristAlois Alzheimer in 1907, is a progressive neurological disorder thatbegins with short term memory loss and proceeds to disorientation,impairment of judgement and reasoning and, ultimately, dementia. Thecourse of the disease usually leads to death in a severely debilitated,immobile state between four and 12 years after onset. AD has beenestimated to afflict 5 to 11 percent of the population over age 65 andas much as 47 percent of the population over age 85. The societal costfor managing AD is upwards of 80 billion dollars annually, primarily dueto the extensive custodial care required for AD patients. Moreover, asadults born during the population boom of the 1940's and 1950's approachthe age when AD becomes more prevalent, the control and treatment of ADwill become an even more significant health care problem. Currently,there is no treatment that significantly retards the progression of thedisease. For reviews on AD, see Selkoe, D. J. Sci. Amer., November 1991,pp. 68-78; and Yankner, B. A. et al. (1991) N. Eng. J. Med.325:1849-1857.

It has recently been reported (Games et al. (1995) Nature 373:523-527)that an Alzheimer-type neuropathology has been created in transgenicmice. The transgenic mice express high levels of human mutant amyloidprecursor protein and progressively develop many of the pathologicalconditions associated with AD.

Pathologically, AD is characterized by the presence of distinctivelesions in the victim's brain. These brain lesions include abnormalintracellular filaments called neurofibrillary tangles (NTFs) andextracellular deposits of amyloidogenic proteins in senile, or amyloid,plaques. Amyloid deposits are also present in the walls of cerebralblood vessels of AD patients. The major protein constituent of amyloidplaques has been identified as a 4 kilodalton peptide called β-amyloidpeptide (β-AP) (Glenner, G. G. and Wong, C. W. (1984) Biochem. Biophys.Res. Commun. 120:885-890; Masters, C. et al. (1985) Proc. Natl. Acad.Sci. USA 82:4245-4249). Diffuse deposits of β-AP are frequently observedin normal adult brains, whereas AD brain tissue is characterized by morecompacted, dense-core β-amyloid plaques. (See e.g., Davies, L. et al.(1988) Neurology 38:1688-1693) These observations occurs in AD. Infurther support of a direct pathogenic role for β-AP, β-amyloid has beenshown to be toxic to mature neurons, both in culture and in vivo.Yankner, B. A. et al. (1989) Science 245:417-420; Yankner, B. A. et al.(1990) Proc. Natl. Acad. Sci. USA 87:9020-9023; Roher, A. E. et al.(1991) Biochem. Biophys. Res. Commun. 174:572-579; Kowall, N. W. et al.(1991) Proc. Natl. Acad. Sci. USA 88:7247-7251. Furthermore, patientswith hereditary cerebral hemorrhage with amyloidosis-Dutch-type(HCHWA-D), which is characterized by diffuse β-amyloid deposits withinthe cerebral cortex and cerebrovasculature, have been shown to have apoint mutation that leads to an amino acid substitution within β-AP.Levy, E. et al. (1990) Science 248:1124-1126. This observationdemonstrates that a specific alteration of the β-AP sequence can causeβ-amyloid to be deposited.

Natural β-AP is derived by proteolysis from a much larger protein calledthe amyloid precursor protein (APP). Kang, J. et al. (1987) Nature325:733; Goldgaber, D. et al. (1987) Science 235:877; Robakis, N. K. etal. (1987) Proc. Natl. Acad. Sci. USA 84:4190; Tanzi, R. E. et al.(1987) Science 235:880. The APP gene maps to chromosome 21, therebyproviding an explanation for the β-amyloid deposition seen at an earlyage in individuals with Down's syndrome, which is caused by trisomy ofchromosome 21. Mann, D. M. et al. (1989) Neuropathol. Appl. Neurobiol.15:317; Rumble, B. et al. (1989) N. Eng. J. Med. 320:1446. APP containsa single membrane spanning domain, with a long amino terminal region(about two-thirds of the protein) extending into the extracellularenvironment and a shorter carboxy-terminal region projecting into thecytoplasm. Differential splicing of the APP messenger RNA leads to atleast five forms of APP, composed of either 563 amino acids (APP-563),695 amino acids (APP-695), 714 amino acids (APP-714), 751 amino acids(APP-751) or 770 amino acids (APP-770).

Within APP, naturally-occurring β amyloid peptide begins at an asparticacid residue at amino acid position 672 of APP-770. Naturally-occurringβ-AP derived from proteolysis of APP is 39 to 43 amino acid residues inlength, depending on the carboxy-terminal end point, which exhibitsheterogeneity. The predominant circulating form of β-AP in the blood andcerebrospinal fluid of both AD patients and normal adults is β1-40(“short β”). Seubert, P. et al. (1992) Nature 359:325; Shoji, M. et al.(1992) Science 258:126. However, β1-42 and β1-43 (“long β”) also areforms in β-amyloid plaques. Masters, C. et al. (1985) Proc. Natl. Acad.Sci. USA 82:4245; Miller, D. et al. (1993) Arch. Biochem. Biophys.301:41; Mori, H. et al. (1992) J. Biol. Chem. 267:17082. Although theprecise molecular mechanism leading to β-APP aggregation and depositionis unknown, the process has been likened to that of nucleation-dependentpolymerizations, such as protein crystallization, microtubule formationand actin polymerization. See e.g., Jarrett, J. T. and Lansbury, P. T.(1993) Cell 73:1055-1058. In such processes, polymerization of monomercomponents does not occur until nucleus formation. Thus, these processesare characterized by a lag time before aggregation occurs, followed byrapid polymerization after nucleation. Nucleation can be accelerated bythe addition of a “seed” or preformed nucleus, which results in rapidpolymerization. The long β forms of β-AP have been shown to act asseeds, thereby accelerating polymerization of both long and short β-APforms. Jarrett, J. T. et al. (1993) Biochemistry 32:4693.

In one study, in which amino acid substitutions were made in β-AP, twomutant β peptides were reported to interfere with polymerization ofnon-mutated β-AP when the mutant and non-mutant forms of peptide weremixed. Hilbich, C. et al. (1992) J. Mol. Biol. 228:460-473. However,equimolar amounts of the mutant and non-mutant (i.e., natural), amyloidpeptides were used to see this effect and the mutant peptides werereported to be unsuitable for use in vivo. Hilbich, C. et al. (1992),supra.

SUMMARY OF THE INVENTION

This invention pertains to compounds, and pharmaceutical compositionsthereof, that can modulate the aggregation of amyloidogenic proteins andpeptides, in particular compounds that can modulate the aggregation ofnatural β amyloid peptides (β-AP) and inhibit the neurotoxicity ofnatural β-APs. In one embodiment, the invention provides an amyloidmodulator compound comprising an amyloidogenic protein, or peptidefragment thereof, coupled directly or indirectly to at least onemodifying group such that the compound modulates the aggregation ofnatural amyloid proteins or peptides when contacted with the naturalamyloidogenic proteins or peptides. Preferably, the compound inhibitsaggregation of natural amyloidogenic proteins or peptides when contactedwith the natural amyloidogenic proteins or peptides. The amyloidogenicprotein, or peptide fragment thereof, can be, for example, selected fromthe group consisting of transthyretin (TTR), prion protein (PrP), isletamyloid polypeptide (IAPP), atrial natriuretic factor (ANF), kappa lightchain, lambda light chain, amyloid A, cystatin C, β2 microglobulin,ApoA-1, gelsolin, procalcitonin, calcitonin, fibrinogen and lysozyme.

In the most preferred embodiment of the invention, the compoundmodulates the aggregation of natural β-AP. The invention provides aβ-amyloid peptide compound comprising a formula:

-   -   wherein Xaa is a β-amyloid peptide having an amino-terminal        amino acid residue corresponding to position 668 of β-amyloid        precursor protein-770 (APP-770) or to a residue carboxy-terminal        to position 668 of APP-770, A is a modifying group attached        directly or indirectly to the β-amyloid peptide of the compound        such that the compound inhibits aggregation of natural β-amyloid        peptides when contacted with the natural β-amyloid peptides, and        n is an integer selected such that the compound inhibits        aggregation of natural β-amyloid peptides when contacted with        the natural β-amyloid peptides.

In one embodiment, at least one A group is attached directly orindirectly to the amino terminus of the β-amyloid peptide of thecompound. In another embodiment, at least one A group is attacheddirectly or indirectly to the carboxy terminus of the β-amyloid peptideof the compound. In yet another embodiment, at least one A group isattached directly or indirectly to a side chain of at least one aminoacid residue of the β-amyloid peptide of the compound.

The invention also provides a β-amyloid modulator compound comprising anAβ aggregation core domain (ACD) coupled directly or indirectly to atleast one modifying group (MG) such that the compound modulates theaggregation or inhibits the neurotoxicity of natural β-amyloid peptideswhen contacted with the natural β-amyloid peptides. Preferably, the Aβaggregation core domain is modeled after a subregion of naturalβ-amyloid peptide between 3 and 10 amino acids in length.

The invention also provides β-amyloid modulator compound comprising aformula:

-   -   wherein Xaa₁, Xaa₂ and Xaa₃ are each amino acid structures and        at least two of Xaa₁, Xaa₂ and Xaa₃ are, independently, selected        from the group consisting of a leucine structure, a        phenylalanine structure and a valine structure;        -   Y, which may or may not be present, is a peptidic structure            having the formula (Xaa)_(a), wherein Xaa is any amino acid            structure and a is an integer from 1 to 15;        -   Z, which may or may not be present, is a peptidic structure            having the formula (Xaa)_(b), wherein Xaa is any amino acid            structure and b is an integer from 1 to 15; and        -   A is a modifying group attached directly or indirectly to            the compound and n is an integer;    -   Xaa₁, Xaa₂, Xaa₃, Y, Z, A and n being selected such that the        compound modulates the aggregation or inhibits the neurotoxicity        of natural β-amyloid peptides when contacted with the natural        β-amyloid peptides. In a preferred embodiment, Xaa₁ and Xaa₂ are        each phenylalanine structures. In another preferred embodiment        Xaa₂ and Xaa₃ are each phenylalanine structures.

The invention further provides a β-amyloid modulator compound comprisinga formula:

-   -   wherein        -   Xaa₁ and Xaa₃ are amino acid structures;        -   Xaa₂ is a valine structure;        -   Xaa₄ is a phenylalanine structure;        -   Y, which may or may not be present, is a peptidic structure            having the formula (Xaa)_(a), wherein Xaa is any amino acid            structure and a is an integer from 1 to 15;        -   Z, which may or may not be present, is a peptidic structure            having the formula (Xaa)_(b), wherein Xaa is any amino acid            structure and b is an integer from 1 to 15; and        -   A is a modifying group attached directly or indirectly to            the compound and n is an integer;    -   Xaa₁, Xaa₃, Y, Z, A and n being selected such that the compound        modulates the aggregation or inhibits the neurotoxicity of        natural β-amyloid peptides when contacted with the natural        β-amyloid peptides. In a preferred embodiment, Xaa₁ is a leucine        structure and Xaa₃ is phenylalanine structure.

The invention still further provides a compound comprising the formula:A—Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈—B

-   -   wherein        -   Xaa1 is a histidine structure;        -   Xaa2 is a glutamine structure;        -   Xaa3 is a lysine structure;        -   Xaa4 is a leucine structure;        -   Xaa5 is a valine structure;        -   Xaa6 is a phenylalanine structure;        -   Xaa7 is a phenylalanine structure;        -   Xaa8 is an alanine structure;        -   A and B are modifying groups attached directly or indirectly            to the amino terminus and carboxy terminus, respectively, of            the compound;    -   and wherein Xaa₁-Xaa₂-Xaa₃, Xaa₁-Xaa₂ or Xaa₁ may or may not be        present;        -   Xaa₈ may or may not be present; and        -   at least one of A and B is present.

The invention still further provides a β-amyloid modulator compoundcomprising a modifying group attached directly or indirectly to apeptidic structure, wherein the peptidic structure comprises amino acidstructures having an amino acid sequence selected from the groupconsisting of His-Gln-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: 5),His-Gln-Lys-Leu-Val-Phe-Phe (SEQ ID NO: 6), Gln-Lys-Leu-Val-Phe-Phe-Ala(SEQ ID NO: 7), Gln-Lys-Leu-Val-Phe-Phe (SEQ ID NO: 8),Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: 9), Lys-Leu-Val-Phe-Phe (SEQ ID NO:10), Leu-Val-Phe-Phe-Ala (SEQ ID NO: 11), Leu-Val-Phe-Phe (SEQ ID NO:12), Leu-Ala-Phe-Phe-Ala (SEQ ID NO: 13), Val-Phe-Phe (SEQ ID NO: 19),Phe-Phe-Ala (SEQ ID NO: 20), Phe-Phe-Val-Leu-Ala (SEQ ID NO: 21),Leu-Val-Phe-Phe-Lys (SEQ ID NO: 22), Leu-Val-Iodotyrosine-Phe-Ala (SEQID NO: 23), Val-Phe-Phe-Ala (SEQ ID NO: 24), Ala-Val-Phe-Phe-Ala (SEQ IDNO: 25), Leu-Val-Phe-Iodotyrosine-Ala (SEQ ID NO: 26),Leu-Val-Phe-Phe-Ala-Glu (SEQ ID NO: 27), Phe-Phe-Val-Leu (SEQ ID NO:28), Phe-Lys-Phe-Val-Leu (SEQ ID NO: 29), Lys-Leu-Val-Ala-Phe (SEQ IDNO: 30), Lys-Leu-Val-Phe-Phe-βAla (SEQ ID NO: 31) andLeu-Val-Phe-Phe-DAla (SEQ ID NO: 32).

In the compounds of the invention comprising a modifying group,preferably the modifying group comprises a cyclic, heterocyclic orpolycyclic group. Preferred modifying groups contains a cis-decalingroup, such as a cholanoyl structure. Preferred modifying groups includea cholyl group, a biotin-containing group, adiethylene-triaminepentaacetyl group, a (−)-menthoxyacetyl group, afluorescein-containing group or an N-acetylneuraminyl group.

The compounds of the invention can be further modified, for example toalter a pharmacokinetic property of the compound or to label thecompound with a detectable substance. Preferred radioactive labels areradioactive iodine or technetium.

The invention also provides a β-amyloid modulator which inhibitsaggregation of natural β-amyloid peptides when contacted with a molarexcess amount of natural β-amyloid peptides.

The invention also provides a β-amyloid peptide compound comprising anamino acid sequence having at least one amino acid deletion compared toβAP₁₋₃₉, such that the compound inhibits aggregation of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides.In one embodiment, the compound has at least one internal amino aciddeleted compared to βAP₁₋₃₉. In another embodiment, the compound has atleast one N-terminal amino acid deleted compared to βAP₁₋₃₉. In yetanother embodiment, the compound has at least one C-terminal amino aciddeleted compared to βAP₁₋₃₉. Preferred compounds include βAP₆₋₂₀ (SEQ IDNO:4), βAP₁₆₋₃₀ (SEQ ID NO: 14), βAP_(1-20, 26-40) (SEQ ID NO: 15) andEEVVHHHHQQ-βAP₁₆₋₄₀ (SEQ ID NO: 16).

The compounds of the invention can be formulated into pharmaceuticalcompositions comprising the compound and a pharmaceutically acceptablecarrier. The compounds can also be used in the manufacture of amedicament for the diagnosis or treatment of an amyloidogenic disease.

Another aspect of the invention pertains to diagnostic and treatmentmethods using the compounds of the invention. The invention provides amethod for inhibiting aggregation of natural β-amyloid peptides,comprising contacting the natural β-amyloid peptides with a compound ofthe invention such that aggregation of the natural β-amyloid peptides isinhibited. The invention also provides a method for inhibitingneurotoxicity of natural β-amyloid peptides, comprising contacting thenatural β-amyloid peptides with a compound of the invention such thatneurotoxicity of the natural β-amyloid peptides is inhibited.

In another embodiment, the invention provides a method for detecting thepresence or absence of natural β-amyloid peptides in a biologicalsample, comprising contacting a biological sample with a compound of theinvention and detecting the compound bound to natural β-amyloid peptidesto thereby detect the presence or absence of natural β-amyloid peptidesin the biological sample. In one embodiment, the β-amyloid modulatorcompound and the biological sample are contacted in vitro. In anotherembodiment, the β-amyloid modulator compound is contacted with thebiological sample by administering the β-amyloid modulator compound to asubject. For in vivo administration, preferably the compound is labeledwith radioactive technetium or radioactive iodine.

In another embodiment, the invention provides a method for detectingnatural β-amyloid peptides to facilitate diagnosis of a β-amyloidogenicdisease, comprising contacting a biological sample with a compound ofthe invention and detecting the compound bound to natural β-amyloidpeptides to facilitate diagnosis of a β-amyloidogenic disease. In oneembodiment, the β-amyloid modulator compound and the biological sampleare contacted in vitro. In another embodiment, the β-amyloid modulatorcompound is contacted with the biological sample by administering theβ-amyloid modulator compound to a subject. For in vivo administration,preferably the compound is labeled with radioactive technetium orradioactive iodine. Preferably, the method facilitates diagnosis ofAlzheimer's disease.

The invention also provides a method for treating a subject for adisorder associated with amyloidosis, comprising administering to thesubject a therapeutically or prophylactically effective amount of acompound of the invention such that the subject is treated for adisorder associated with amyloidosis. The method can be used to treatdisorders is selected, for example, from the group consisting offamilial amyloid polyneuropathy (Portuguese, Japanese and Swedishtypes), familial amyloid cardiomyopathy (Danish type), isolated cardiacamyloid, systemic senile amyloidosis, scrapie, bovine spongiformencephalopathy, Creutzfeldt-Jakob disease,Gerstmann-Straussler-Scheinker syndrome, adult onset diabetes,insulinoma, isolated atrial amyloidosis, idiopathic (primary)amyloidosis, myeloma or macroglobulinemia-associated amyloidosis,primary localized cutaneous nodular amyloidosis associated withSjogren's syndrome, reactive (secondary) amyloidosis, familialMediterranean Fever and familial amyloid nephropathy with urticaria anddeafness (Muckle-Wells syndrome), hereditary cerebral hemorrhage withamyloidosis of Icelandic type, amyloidosis associated with long termhemodialysis, hereditary non-neuropathic systemic amyloidosis (familialamyloid polyneuropathy M), familial amyloidosis of Finnish type,amyloidosis associated with medullary carcinoma of the thyroid,fibrinogen-associated hereditary renal amyloidosis andlysozyme-associated hereditary systemic amyloidosis.

In a preferred embodiment, the invention provides a method for treatinga subject for a disorder associated with β-amyloidosis, comprisingadministering to the subject a therapeutically or prophylacticallyeffective amount of a compound of the invention such that the subject istreated for a disorder associated with β-amyloidosis. Preferably thedisorder is Alzheimer's disease.

In yet another embodiment, the invention provides a method for treatinga subject for a disorder associated with β-amyloidosis, comprisingadministering to the subject a recombinant expression vector encoding apeptide compound of the invention such that the compound is synthesizedin the subject and the subject is treated for a disorder associated withβ-amyloidosis. Preferably, the disorder is Alzheimer's disease.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graphic representation of the turbidity of a β-AP₁₋₄₀solution, as measured by optical density at 400 nm, either in theabsence of a β-amyloid modulator or in the presence of the β-amyloidmodulator N-biotinyl-βAP₁₋₄₀ (1%, or 5%).

FIG. 2 is a schematic representation of compounds which can be used tomodify a β-AP or an Aβ aggregation core domain to form a β-amyloidmodulator of the invention.

FIG. 3 is a graphic representation of the toxicity of Aβ₁₋₄₀ aggregates,but not Aβ₁₋₄₀ monomers, to cultured neuronal cells.

FIG. 4 is a graphic representation of the aggregation of Aβ₁₋₄₀ in thepresence of an equimolar amount of cholyl-Aβ₆₋₂₀ (panel A), a ˜2-foldmolar excess of cholyl-Aβ₆₋₂₀ (panel B) or a ˜6-fold molar excess ofcholyl-Aβ₆₋₂₀ (panel C) and the corresponding toxicity of the aggregatesof panels A, B and C to cultured neuronal cells (panels D, E and F,respectively).

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to compounds, and pharmaceutical compositionsthereof, that can modulate the aggregation of amyloidogenic proteins andpeptides, in particular compounds that can modulate the aggregation ofnatural β amyloid peptides (β-AP) and inhibit the neurotoxicity ofnatural β-APs. A compound of the invention that modulates aggregation ofnatural β-AP, referred to herein interchangeably as a β amyloidmodulator compound, a β amyloid modulator or simply a modulator, altersthe aggregation of natural β-AP when the modulator is contacted withnatural β-AP. Thus, a compound of the invention acts to alter thenatural aggregation process or rate for β-AP, thereby disrupting thisprocess. Preferably, the compounds inhibit β-AP aggregation.Furthermore, the invention provides subregions of the β amyloid peptidethat are sufficient, when appropriately modified as described herein, toalter (and preferably inhibit) aggregation of natural β amyloid peptideswhen contacted with the natural β amyloid peptides. In particular,preferred modulator compounds of the invention are comprised of amodified form of an Aβ aggregation core domain, modeled after theaforementioned Aβ subregion (as described further below), which issufficient to alter (and preferably inhibit) the natural aggregationprocess or rate for β-AP. This Aβ aggregation core domain can comprisesas few as three amino acid residues (or derivative, analogues ormimetics thereof). Moreover, while the amino acid sequence of the Aβaggregation core domain can directly correspond to an amino acidsequence found in natural β-AP, it is not essential that the amino acidsequence directly correspond to a β-AP sequence. Rather, amino acidresidues derived from a preferred subregion of β-AP (a hydrophobicregion centered around positions 17-20) can be rearranged in orderand/or substituted with homologous residues within a modulator compoundof the invention and yet maintain their inhibitory activity (describedfurther below).

The β amyloid modulator compounds of the invention can be selected basedupon their ability to inhibit the aggregation of natural β-AP in vitroand/or inhibit the neurotoxicity of natural β-AP fibrils for culturedcells (using assays described herein). Accordingly, the preferredmodulator compounds inhibit the aggregation of natural β-AP and/orinhibit the neurotoxicity of natural β-AP. However, modulator compoundsselected based on one or both of these properties may have additionalproperties in vivo that may be beneficial in the treatment ofamyloidosis. For example, the modulator compound may interfere withprocessing of natural β-AP (either by direct or indirect proteaseinhibition) or by modulation of processes that produce toxic β-AP, orother APP fragments, in vivo. Alternatively, modulator compounds may beselected based on these latter properties, rather than inhibition of Aβaggregation in vitro. Moreover, modulator compounds of the inventionthat are selected based upon their interaction with natural β-AP alsomay interact with APP or with other APP fragments.

As used herein, a “modulator” of β-amyloid aggregation is intended torefer to an agent that, when contacted with natural β amyloid peptides,alters the aggregation of the natural β amyloid peptides. The term“aggregation of β amyloid peptides” refers to a process whereby thepeptides associate with each other to form a multimeric, largelyinsoluble complex. The term “aggregation” further is intended toencompass β amyloid fibril formation and also encompasses β-amyloidplaques.

The terms “natural β-amyloid peptide”, “natural β-AP” and “natural Aβpeptide”, used interchangeably herein, are intended to encompassnaturally occurring proteolytic cleavage products of the β amyloidprecursor protein (APP) which are involved in β-AP aggregation andβ-amyloidosis. These natural peptides include β-amyloid peptides having39-43 amino acids (i.e., Aβ₁₋₃₉, Aβ₁₋₄₀, Aβ₁₋₄₁, Aβ₁₋₄₂ and Aβ₁₋₄₃). Theamino-terminal amino acid residue of natural β-AP corresponds to theaspartic acid residue at position 672 of the 770 amino acid residue formof the amyloid precursor protein (“APP-770”). The 43 amino acid longform of natural β-AP has the amino acid sequence

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT

(also shown in SEQ ID NO: 1), whereas the shorter forms have 1-4 aminoacid residues deleted from the carboxy-terminal end. The amino acidsequence of APP-770 from position 672 (i.e., the amino-terminus ofnatural β-AP) to its C-terminal end (103 amino acids) is shown in SEQ IDNO: 2. The preferred form of natural β-AP for use in the aggregationassays described herein is Aβ₁₋₄₀.

In the presence of a modulator of the invention, aggregation of naturalβ amyloid peptides is “altered” or “modulated”. The various forms of theterm “alteration” or “modulation” are intended to encompass bothinhibition of β-AP aggregation and promotion of β-AP aggregation.Aggregation of natural β-AP is “inhibited” in the presence of themodulator when there is a decrease in the amount and/or rate of β-APaggregation as compared to the amount and/or rate of β-AP aggregation inthe absence of the modulator. The various forms of the term “inhibition”are intended to include both complete and partial inhibition of β-APaggregation. Inhibition of aggregation can be quantitated as the foldincrease in the lag time for aggregation or as the decrease in theoverall plateau level of aggregation (i.e., total amount ofaggregation), using an aggregation assay as described in the Examples.In various embodiments, a modulator of the invention increases the lagtime of aggregation at least 1.2-fold, 1.5-fold, 1.8-fold, 2-fold,2.5-fold, 3-fold, 4-fold or 5-fold. In various other embodiments, amodulator of the invention inhibits the plateau level of aggregation atleast 10%, 20%, 30%, 40%, 50%, 75% or 100%.

A modulator which inhibits β-AP aggregation (an “inhibitory modulatorcompound”) can be used to prevent or delay the onset of β-amyloiddeposition. Moreover, as demonstrated in Example 10, inhibitorymodulator compounds of the invention inhibit the formation and/oractivity of neurotoxic aggregates of natural Aβ peptide (i.e., theinhibitory compounds can be used to inhibit the neurotoxicity of β-AP).Still further, also as demonstrated in Example 10, the inhibitorycompounds of the invention can be used to reduce the neurotoxicity ofpreformed β-AP aggregates, indicating that the inhibitory modulators caneither bind to preformed Aβ fibrils or soluble aggregate and modulatetheir inherent neurotoxicity or that the modulators can perturb theequilibrium between monomeric and aggregated forms of β-AP in favor ofthe non-neurotoxic form.

Alternatively, in another embodiment, a modulator compound of theinvention promotes the aggregation of natural Aβ peptides. The variousforms of the term “promotion” refer to an increase in the amount and/orrate of β-AP aggregation in the presence of the modulator, as comparedto the amount and/or rate of β-AP aggregation in the absence of themodulator. Such a compound which promotes Aβ aggregation is referred toas a stimulatory modulator compound. Stimulatory modulator compounds maybe useful for sequestering β-amyloid peptides, for example in abiological compartment where aggregation of β-AP may not be deleteriousto thereby deplete β-AP from a biological compartment where aggregationof β-AP is deleterious. Moreover, stimulatory modulator compounds can beused to promote Aβ aggregation in in vitro aggregation assays (e.g.,assays such as those described in the Examples), for example inscreening assays for test compounds that can then inhibit or reversethis Aβ aggregation (i.e., a stimulatory modulator compound can act as a“seed” to promote the formation of Aβ aggregates).

In a preferred embodiment, the modulators of the invention are capableof altering β-AP aggregation when contacted with a molar excess amountof natural β-AP. A “molar excess amount of natural β-AP” refers to aconcentration of natural β-AP, in moles, that is greater than theconcentration, in moles, of the modulator. For example, if the modulatorand β-AP are both present at a concentration of 1 μM, they are said tobe “equimolar”, whereas if the modulator is present at a concentrationof 1 μM and the β-AP is present at a concentration of 5 μM, the β-AP issaid to be present at a 5-fold molar excess amount compared to themodulator. In preferred embodiments, a modulator of the invention iseffective at altering natural β-AP aggregation when the natural β-AP ispresent at at least a 2-fold, 3-fold or 5-fold molar excess compared tothe concentration of the modulator. In other embodiments, the modulatoris effective at altering β-AP aggregation when the natural β-AP ispresent at at least a 10-fold, 20-fold, 33-fold, 50-fold, 100-fold,500-fold or 1000-fold molar excess compared to the concentration of themodulator.

Various additional aspects of the modulators of the invention, and theuses thereof, are described in further detail in the followingsubsections.

I. Modulator Compounds

In one embodiment, a modulator of the invention comprises a β-amyloidpeptide compound comprising the formula:

-   -   wherein Xaa is a β-amyloid peptide, A is a modulating group        attached directly or indirectly to the β-amyloid peptide of the        compound such that the compound inhibits aggregation of natural        β-amyloid peptides when contacted with the natural β-amyloid        peptides, and n is an integer selected such that the compound        inhibits aggregation of natural β-amyloid peptides when        contacted with the natural β-amyloid peptides.

Preferably, β-amyloid peptide of the compound has an amino-terminalamino acid residue corresponding to position 668 of β-amyloid precursorprotein-770 (APP-770) or to a residue carboxy-terminal to position 668of APP-770. The amino acid sequence of APP-770 from position 668 toposition 770 (i.e., the carboxy terminus) is shown below and in SEQ IDNO: 2:

EVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQ MQNMore preferably, the amino-terminal amino acid residue of the β-amyloidpeptide corresponds to position 672 of APP-770 (position 5 of the aminoacid sequence of SEQ ID NO: 2) or to a residue carboxy-terminal toposition 672 of APP-770. Although the β-amyloid peptide of the compoundmay encompass the 103 amino acid residues corresponding to positions668-770 of APP-770, preferably the peptide is between 6 and 60 aminoacids in length, more preferably between 10 and 43 amino acids in lengthand even more preferably between 10 and 25 amino acid residues inlength.

As used herein, the term “β amyloid peptide”, as used in a modulator ofthe invention is intended to encompass peptides having an amino acidsequence identical to that of the natural sequence in APP, as well aspeptides having acceptable amino acid substitutions from the naturalsequence. Acceptable amino acid substitutions are those that do notaffect the ability of the peptide to alter natural β-AP aggregation.Moreover, particular amino acid substitutions may further contribute tothe ability of the peptide to alter natural β-AP aggregation and/or mayconfer additional beneficial properties on the peptide (e.g., increasedsolubility, reduced association with other amyloid proteins, etc.). Forexample, substitution of hydrophobic amino acid residues for the twophenylalanine residues at positions 19 and 20 of natural β-AP (positions19 and 20 of the amino acid sequence shown in SEQ ID NO: 1) may furthercontribute to the ability of the peptide to alter β-AP aggregation (seeHilbich, C. (1992) J. Mol. Biol. 228:460-473). Thus, in one embodiment,the M-AP of the compound consists of the amino acid sequence shown belowand in SEQ ID NO: 3:

DAEFRHDSGYEVHHQKLV(Xaa₁₉)(Xaa₂₀)AEDVGSNKGAIIGLMVGGVVIAT

(or an amino-terminal or carboxy-terminal deletion thereof), wherein Xaais a hydrophobic amino acid. Examples of hydrophobic amino acids areisoleucine, leucine, threonine, serine, alanine, valine or glycine.Preferably, F₁₉F₂₀ is substituted with T₁₉T₂₀ or G₁₉I₂₀.

Other suitable amino acid substitutions include replacement of aminoacids in the human peptide with the corresponding amino acids of therodent β-AP peptide. The three amino acid residues that differ betweenhuman and rat β-AP are at positions 5, 10 and 13 of the amino acidsequence shown in SEQ ID NOs: 1 and 3. A human β-AP having the human torodent substitutions Arg₅ to Gly, Tyr₁₀ to Phe and His₁₃ to Arg has beenshown to retain the properties of the human peptide (see Fraser, P. E.et al. (1992) Biochemistry 31:10716-10723; and Hilbich, C. et al. (1991)Eur. J. Biochem. 201:61-69). Accordingly, a human β-AP having rodentβ-AP a.a. substitutions is suitable for use in a modulator of theinvention.

Other possible β-AP amino acid substitutions are described in Hilbich,C. et al. (1991) J. Mol. Biol. 218:149-163; and Hilbich, C. (1992) J.Mol. Biol. 228:460-473. Moreover, amino acid substitutions that affectthe ability of β-AP to associate with other proteins can be introduced.For example, one or more amino acid substitutions that reduce theability of β-AP to associate with the serpin enzyme complex (SEC)receptor, α1-antichymotrypsin (ACT) and/or apolipoprotein E (ApoE) canbe introduced. A preferred substitution for reducing binding to the SECreceptor is L₃₄M₃₅ to A₃₄A₃₅ (at positions 34 and 35 of the amino acidsequences shown in SEQ ID NOs: 1 and 3). A preferred substitution forreducing binding to ACT is S₈ to A₈ (at position 8 of the amino acidsequences shown in SEQ ID NOs: 1 and 3).

Alternative to β-AP amino acid substitutions described herein or knownin the art, a modulator composed, at least in part, of an aminoacid-substituted β amyloid peptide can be prepared by standardtechniques and tested for the ability to alter β-AP aggregation using anaggregation assay described herein. To retain the properties of theoriginal modulator, preferably conservative amino acid substitutions aremade at one or more amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art,including basic side chains (e.g., lysine, arginine, histidine), acidicside chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),β-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Accordingly, a modulator composed of a β amyloid peptidehaving an amino acid sequence that is mutated from that of the wild-typesequence in APP-770 yet which still retains the ability to alter naturalβ-AP aggregation is within the scope of the invention.

As used herein, the term “β amyloid peptide” is further intended toinclude peptide analogues or peptide derivatives or peptidomimetics thatretain the ability to alter natural β-AP aggregation as describedherein. For example, a β amyloid peptide of a modulator of the inventionmay be modified to increase its stability, bioavailability, solubility,etc. The terms “peptide analogue”, “peptide derivative” and“peptidomimetic” as used herein are intended to include molecules whichmimic the chemical structure of a peptide and retain the functionalproperties of the peptide. Approaches to designing peptide analogs areknown in the art. For example, see Farmer, P. S. in Drug Design (E. J.Ariens, ed.) Academic Press, New York, 1980, vol. 10, pp. 119-143; Ball.J. B. and Alewood, P. F. (1990) J. Mol. Recognition 3:55; Morgan, B. A.and Gainor, J. A. (1989) Ann. Rep. Med. Chem. 24:243; and Freidinger, R.M. (1989) Trends Pharmacol. Sci. 10:270. Examples of peptide analogues,derivatives and peptidomimetics include peptides substituted with one ormore benzodiazepine molecules (see e.g., James, G. L. et al. (1993)Science 260:1937-1942), peptides with methylated amide linkages and“retro-inverso” peptides (see U.S. Pat. No. 4,522,752 by Sisto). Peptideanalogues, peptide derivatives and peptidomimetic are described infurther detail below with regard to compounds comprising an Aβaggregation core domain.

In a modulator of the invention having the formula shown above, amodulating group (“A”) is attached directly or indirectly to theβ-amyloid peptide of the modulator (As used herein, the term “modulatinggroup” and “modifying group” are used interchangeably to describe achemical group directly or indirectly attached to an Aβ derived peptidicstructure). For example, the modulating group can be directly attachedby covalent coupling to the β-amyloid peptide or the modulating groupcan be attached indirectly by a stable non-covalent association. In oneembodiment of the invention, the modulating group is attached to theamino-terminus of the β-amyloid peptide of the modulator. Accordingly,the modulator can comprise a compound having a formula:

Alternatively, in another embodiment of the invention, the modulatinggroup is attached to the carboxy-terminus of the β-amyloid peptide ofthe modulator. Accordingly, the modulator can comprise a compound havinga formula:

In yet another embodiment, the modulating group is attached to the sidechain of at least one amino acid residue of the β-amyloid peptide of thecompound (e.g., through the epsilon amino group of a lysyl residue(s),through the carboxyl group of an aspartic acid residue(s) or a glutamicacid residue(s), through a hydroxy group of a tyrosyl residue(s), aserine residue(s) or a threonine residue(s) or other suitable reactivegroup on an amino acid side chain).

The modulating group is selected such that the compound inhibitsaggregation of natural β-amyloid peptides when contacted with thenatural β-amyloid peptides. Accordingly, since the β-AP peptide of thecompound is modified from its natural state, the modulating group “A” asused herein is not intended to include hydrogen. In a preferredembodiment, the modulating group is a biotin compound of the formula:

-   -   wherein X₁-X₃ are each independently selected from the group        consisting of S, O and NR₂, wherein R₂ is hydrogen, or an aryl,        lower alkyl, alkenyl or alkynyl moiety; W is ═O or N(R₂)₂; R₁ is        a lower alkylenyl moiety and Y is a direct bond or a spacer        molecule selected for its ability to react with a target group        on a β-AP. At least one of X₁-X₃ is an NR₂ group or W m is an        N(R₁)₂ group.

The term “aryl” is intended to include aromatic moieties containingsubstituted or unsubstituted ring(s), e.g., benzyl, naphthyl etc. Othermore complex fused ring moieties also are intended to be included.

The term “lower alkyl or alkylenyl moiety” refers to a saturated,straight or branched chain (or combination thereof) hydrocarboncontaining 1 to about 6 carbon atoms, more preferably from 1 to 3 carbonatoms. The terms “lower alkenyl moiety” and “lower alkynyl moiety” referto unsaturated hydrocarbons containing 1 to about 6 carbon atoms, morepreferably 1 to 3 carbon atoms. Preferably, R₂ contains 1 to 3 carbonatoms. Preferably, R₁ contains 4 carbon atoms.

The spacer molecule (Y) can be, for example, a lower alkyl group or alinker peptide, and is preferably selected for its ability to link witha free amino group (e.g., the α-amino group at the amino-terminus of aβ-AP). Thus, in a preferred embodiment, the biotin compound modifies theamino-terminus of a β-amyloid peptide.

Additional suitable modulating groups may include other cyclic andheterocyclic compounds and other compounds having similar steric “bulk”.Non-limiting examples of compounds which can be used to modify a β-APare shown schematically in FIG. 2, and include N-acetylneuraminic acid,cholic acid, trans-4-cotininecarboxylic acid,2-imino-1-imidazolidineacetic acid, (S)-(−)-indoline-2-carboxylic acid,(−)-menthoxyacetic acid, 2-norbornaneacetic acid,γ-oxo-5-acenaphthenebutyric acid, (−)-2-oxo-4-thiazolidinecarboxylicacid, tetrahydro-3-furoic acid, 2-iminobiotin-N-hydroxysuccinimideester, diethylenetriaminepentaacetic dianhydride, 4-morpholinecarbonylchloride, 2-thiopheneacetyl chloride, 2-thiophenesulfonyl chloride,5-(and 6-)-carboxyfluorescein (succinimidyl ester), fluoresceinisothiocyanate, and acetic acid (or derivatives thereof). Suitablemodulating groups are described further in subsection II below.

In a modulator of the invention, a single modulating group may beattached to a amyloid peptide (e.g., n=1 in the formula shown above) ormultiple modulating groups may be attached to the peptide. The number ofmodulating groups is selected such that the compound inhibitsaggregation of natural β-amyloid peptides when contacted with thenatural β-amyloid peptides. However, n preferably is an integer between1 and 60, more preferably between 1 and 30 and even more preferablybetween 1 and 10 or 1 and 5.

In another embodiment, a β-amyloid modulator compound of the inventioncomprises an Aβ aggregation core domain (abbreviated as ACD) coupleddirectly or indirectly to a modifying group such that the compoundmodulates the aggregation or inhibits the neurotoxicity of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides.As used herein, an “Aβ aggregation core domain” is intended to refer toa structure that is modeled after a subregion of a natural β-amyloidpeptide which is sufficient to modulate aggregation of natural β-APswhen this subregion of the natural G-AP is appropriately modified asdescribed herein (e.g., modified at the amino-terminus). The term“subregion of a natural β-amyloid peptide” is intended to includeamino-terminal and/or carboxy-terminal deletions of natural G-AP. Theterm “subregion of natural β-AP” is not intended to include full-lengthnatural G-AP (i.e., “subregion” does not include Aβ₁₋₃₉, Aβ₁₋₄₀, Aβ₁₋₄₁,Aβ₁₋₄₂ and Aβ₁₋₄₃).

Although not intending to be limited by mechanism, the ACD of themodulators of the invention is thought to confer a specific targetingfunction on the compound that allows the compound to recognize andspecifically interact with natural β-AP. Preferably, the ACD is modeledafter a subregion of natural β-AP that is less than 15 amino acids inlength and more preferably is between 3-10 amino acids in length. Invarious embodiments, the ACD is modeled after a subregion of β-AP thatis 10, 9, 8, 7, 6, 5, 4 or 3 amino acids in length. In one embodiment,the subregion of β-AP upon which the ACD is modeled is an internal orcarboxy-terminal region of β-AP (i.e., downstream of the amino-terminusat amino acid position 1). In another embodiment, the ACD is modeledafter a subregion of β-AP that is hydrophobic. In certain specificembodiments, the term Aβ aggregation core domain specifically excludesβ-AP subregions corresponding to amino acid positions 1-15 (Aβ₁₋₁₅),6-20 (Aβ₆₋₂₀) and 16-40 (Aβ₁₆₋₄₀).

An Aβ aggregation core domain can be comprised of amino acid residueslinked by peptide bonds. That is, the ACD can be a peptide correspondingto a subregion of β-AP. Alternatively, an Aβ aggregation core domain canbe modeled after the natural Aβ peptide region but may be comprised of apeptide analogue, peptide derivative or peptidomimetic compound, orother similar compounds which mimics the structure and function of thenatural peptide. Accordingly, as used herein, an “Aβ aggregation coredomain” is intended to include peptides, peptide analogues, peptidederivatives and peptidomimetic compounds which, when appropriatelymodified, retain the aggregation modulatory activity of the modifiednatural Aβ peptide subregion. Such structures that are designed basedupon the amino acid sequence are referred to herein as “Aβ derivedpeptidic structures.” Approaches to designing peptide analogues,derivatives and mimetics are known in the art. For example, see Farmer,P. S. in Drug Design (E. J. Ariens, ed.) Academic Press, New York, 1980,vol. 10, pp. 119-143; Ball. J. B. and Alewood, P. F. (1990) J. Mol.Recognition 3:55; Morgan, B. A. and Gainor, J. A. (1989) Ann. Rep. Med.Chem. 24:243; and Freidinger, R. M. (1989) Trends Pharmacol. Sci.10:270. See also Sawyer, T. K. (1995) “Peptidomimetic Design andChemical Approaches to Peptide Metabolism” in Taylor, M. D. and Amidon,G. L. (eds.) Peptide-Based Drug Design: Controlling Transport andMetabolism, Chapter 17; Smith, A. B. 3rd, et al. (1995) J. Am. Chem.Soc. 117:11113-11123; Smith, A. B. 3rd, et al. (1994) J. Am. Chem. Soc.116:9947-9962; and Hirschman, R., et al. (1993) J. Am. Chem. Soc.115:12550-12568.

As used herein, a “derivative” of a compound X (e.g., a peptide or aminoacid) refers to a form of X in which one or more reaction groups on thecompound have been derivatized with a substituent group. Examples ofpeptide derivatives include peptides in which an amino acid side chain,the peptide backbone, or the amino- or carboxy-terminus has beenderivatized (e.g., peptidic compounds with methylated amide linkages).As used herein an “analogue” of a compound X refers to a compound whichretains chemical structures of X necessary for functional activity of Xyet which also contains certain chemical structures which differ from X.An examples of an analogue of a naturally-occurring peptide is apeptides which includes one or more non-naturally-occurring amino acids.As used herein, a “mimetic” of a compound X refers to a compound inwhich chemical structures of X necessary for functional activity of Xhave been replaced with other chemical structures which mimic theconformation of X. Examples of peptidomimetics include peptidiccompounds in which the peptide backbone is substituted with one or morebenzodiazepine molecules (see e.g., James, G. L. et al. (1993) Science260:1937-1942), peptides in which all L-amino acids are substituted withthe corresponding D-amino acids and “retro-inverso” peptides (see U.S.Pat. No. 4,522,752 by Sisto), described further below.

The term mimetic, and in particular, peptidomimetic, is intended toinclude isosteres. The term “isostere” as used herein is intended toinclude a chemical structure that can be substituted for a secondchemical structure because the steric conformation of the firststructure fits a binding site specific for the second structure. Theterm specifically includes peptide back-bone modifications (i.e., amidebond mimetics) well known to those skilled in the art. Suchmodifications include modifications of the amide nitrogen, the α-carbon,amide carbonyl, complete replacement of the amide bond, extensions,deletions or backbone crosslinks. Several peptide backbone modificationsare known, including ψ[CH₂S], ψ[CH₂NH], ψ[CSNH₂], ψ[NHCO], ψ[COCH₂], andψ[(E) or (Z) CH═CH]. In the nomenclature used above, ψ indicates theabsence of an amide bond. The structure that replaces the amide group isspecified within the brackets. Other examples of isosteres includepeptides substituted with one or more benzodiazepine molecules (seee.g., James, G. L. et al. (1993) Science 260:1937-1942)

Other possible modifications include an N-alkyl (or aryl) substitution(ψ[CONR]), backbone crosslinking to construct lactams and other cyclicstructures, substitution of all D-amino acids for all L-amino acidswithin the compound (“inverso” compounds) or retro-inverso amino acidincorporation (ψ[NHCO]). By “inverso” is meant replacing L-amino acidsof a sequence with D-amino acids, and by “retro-inverso” or“enantio-retro” is meant reversing the sequence of the amino acids(“retro”) and replacing the L-amino acids with D-amino acids. Forexample, if the parent peptide is Thr-Ala-Tyr, the retro modified formis Tyr-Ala-Thr, the inverso form is thr-ala-tyr, and the retro-inversoform is tyr-ala-thr (lower case letters refer to D-amino acids).Compared to the parent peptide, a retro-inverso peptide has a reversedbackbone while retaining substantially the original spatial conformationof the side chains, resulting in a retro-inverso isomer with a topologythat closely resembles the parent peptide. See Goodman et al.“Perspectives in Peptide Chemistry” pp. 283-294 (1981). See also U.S.Pat. No. 4,522,752 by Sisto for further description of “retro-inverso”peptides.

Other derivatives of the modulator compounds of the invention includeC-terminal hydroxymethyl derivatives, O-modified derivatives (e.g.,C-terminal hydroxymethyl benzyl ether), N-terminally modifiedderivatives including substituted amides such as alkylamides andhydrazides and compounds in which a C-terminal phenylalanine residue isreplaced with a phenethylamide analogue (e.g., Val-Phe-phenethylamide asan analogue of the tripeptide Val-Phe-Phe).

In a preferred embodiment, the ACD of the modulator is modeled after thesubregion of β-AP encompassing amino acid positions 17-20 (i.e.,Leu-Val-Phe-Phe; SEQ ID NO: 12). As described further in Examples 7, 8and 9, peptide subregions of Aβ₁₋₄₀ were prepared, amino-terminallymodified and evaluated for their ability to modulate aggregation ofnatural β-amyloid peptides. One subregion that was effective atinhibiting aggregation was Aβ₆₋₂₀ (i.e., amino acid residues 6-20 of thenatural Aβ₁₋₄₀ peptide, the amino acid sequence of which is shown in SEQID NO: 4). Amino acid residues were serially deleted from theamino-terminus or carboxy terminus of this subregion to furtherdelineate a minimal subregion that was sufficient for aggregationinhibitory activity. This process defined Aβ₁₇₋₂₀ (i.e., amino acidresidues 17-20 of the natural Aβ₁₋₄₀ peptide) as a minimal subregionthat, when appropriately modified, is sufficient for aggregationinhibitory activity. Accordingly, an “Aβ aggregation core domain” withina modulator compound of the invention can be modeled after Aβ₁₇₋₂₀. Inone embodiment, the Aβ aggregation core domain comprises Aβ₁₇₋₂₀ itself(i.e., a peptide comprising the amino acid sequenceleucine-valine-phenylalanine-phenylalanine; SEQ ID NO: 12). In otherembodiments, the structure of Aβ₁₇₋₂₀ is used as a model to design an Aβaggregation core domain having similar structure and function toAβ₁₇₋₂₀. For example, peptidomimetics, derivatives or analogues ofAβ₁₇₋₂₀ (as described above) can be used as an Aβ aggregation coredomain. In addition to Aβ₁₇₋₂₀, the natural Aβ peptide is likely tocontain other minimal subregions that are sufficient for aggregationinhibitory activity. Such additional minimal subregions can beidentified by the processes described in Examples 7, 8 and 9, wherein a15mer subregion of Aβ₁₋₄₀ is serially deleted from the amino-terminus orcarboxy terminus, the deleted peptides are appropriately modified andthen evaluated for aggregation inhibitory activity.

One form of the β-amyloid modulator compound comprising an Aβaggregation core domain modeled after Aβ₁₇₋₂₀ coupled directly orindirectly to at least one modifying group has the formula:

-   -   wherein        -   Xaa₁ and Xaa₃ are amino acid structures;        -   Xaa₂ is a valine structure;        -   Xaa₄ is a phenylalanine structure;        -   Y, which may or may not be present, is a peptidic structure            having the formula (Xaa)_(a), wherein Xaa is any amino acid            structure and a is an integer from 1 to 15;        -   Z, which may or may not be present, is a peptidic structure            having the formula (Xaa)_(b), wherein Xaa is any amino acid            structure and b is an integer from 1 to 15; and        -   A is a modifying group attached directly or indirectly to            the compound and n is an integer;    -   Xaa₁, Xaa₃, Y, Z, A and n being selected such that the compound        modulates the aggregation or inhibits the neurotoxicity of        natural β-amyloid peptides when contacted with the natural        β-amyloid peptides.

Preferably, a modulator compound of the above formula inhibitsaggregation of natural β-amyloid peptides when contacted with thenatural β-amyloid peptides and/or inhibits Aβ neurotoxicity.Alternatively, the modulator compound can promote aggregation of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides.The type and number of modifying groups (“A”) coupled to the modulatorare selected such that the compound alters (and preferably inhibits)aggregation of natural β-amyloid peptides when contacted with thenatural β-amyloid peptides. A single modifying group can be coupled tothe modulator (i.e., n=1 in the above formula) or, alternatively,multiple modifying groups can be coupled to the modulator. In variousembodiments, n is an integer between 1 and 60, between 1 and 30, between1 and 10, between 1 and 5 or between 1 and 3. Suitable types ofmodifying groups are described further in subsection II below.

As demonstrated in Example 9, amino acid positions 18 (Val₁₈) and 20(Phe₂₀) of Aβ₁₇₋₂₀ (corresponding to Xaa₂ and Xaa₄) are particularlyimportant within the core domain for inhibitory activity of themodulator compound. Accordingly, these positions are conserved withinthe core domain in the formula shown above. The terms “valine structure”and “phenylalanine structure” as used in the above formula are intendedto include the natural amino acids, as well as non-naturally-occurringanalogues, derivatives and mimetics of valine and phenylalanine,respectively, (including D-amino acids) which maintain the functionalactivity of the compound. Moreover, although Val₁₈ and Phe₂₀ have animportant functional role, it is possible that Xaa₂ and/or Xaa₄ can besubstituted with other naturally-occurring amino acids that arestructurally related to valine or phenylalanine, respectively, whilestill maintaining the activity of the compound. Thus, the terms “valinestructure” is intended to include conservative amino acid substitutionsthat retain the activity of valine at Xaa₂, and the term “phenylalaninestructure” is intended to include conservative amino acid substitutionsthat retain the activity of phenylalanine at Xaa₄. However, the term“valine structure” is not intended to include threonine.

In contrast to positions 18 and 20 of Aβ17-20, a Phe to Ala substitutionat position 19 (corresponding to Xaa₃) did not abolish the activity ofthe modulator, indicating position 19 may be more amenable to amino acidsubstitution. In various embodiments of the above formula, positionsXaa₁ and Xaa₃ are any amino acid structure. The term “amino acidstructure” is intended to include natural and non-natural amino acids aswell as analogues, derivatives and mimetics thereof, including D-aminoacids. In a preferred embodiment of the above formula, Xaa₁ is a leucinestructure and Xaa₃ is a phenylalanine structure (i.e., modeled afterLeu₁₇ and Phe₁₉, respectively, in the natural Aβ peptide sequence). Theterm “leucine structure” is used in the same manner as valine structureand phenylalanine structure described above. Alternatively, an anotherembodiment, Xaa₃ is an alanine structure.

The four amino acid structure ACD of the modulator of the above formulacan be flanked at the amino-terminal side, carboxy-terminal side, orboth, by peptidic structures derived either from the natural Aβ peptidesequence or from non-Aβ sequences. The term “peptidic structure” isintended to include peptide analogues, derivatives and mimetics thereof,as described above. The peptidic structure is composed of one or morelinked amino acid structures, the type and number of which in the aboveformula are variable. For example, in one embodiment, no additionalamino acid structures flank the Xaa₁-Xaa₂-Xaa₃-Xaa₄ core sequence (i.e.,Y and Z are absent in the above formula). In another embodiment, one ormore additional amino acid structures flank only the amino-terminus ofthe core sequences (i.e., Y is present but Z is absent in the aboveformula). In yet another embodiment, one or more additional amino acidstructures flank only the carboxy-terminus of the core sequences (i.e.,Z is present but Y is absent in the above formula). The length offlanking Z or Y sequences also is variable. For example, in oneembodiment, a and b are integers from 1 to 15. More preferably, a and bare integers between 1 and 10. Even more preferably, a and b areintegers between 1 and 5. Most preferably, a and b are integers between1 and 3.

One form of the β-amyloid modulator compound comprising an Aβaggregation core domain modeled after Aβ₁₇₋₂₀ coupled directly orindirectly to at least one modifying group has the formula:A—(Y)—Xaa₁-Xaa₂-Xaa₃-Xaa₄—(Z)—B

-   -   wherein        -   Xaa₁ and Xaa₃ are amino acids or amino acid mimetics;        -   Xaa₂ is valine or a valine mimetic        -   Xaa₄ is phenylalanine or a phenylalanine mimetic;        -   Y, which may or may not be present, is a peptide or            peptidomimetic having the formula (Xaa)_(a), wherein Xaa is            any amino acid or amino acid mimetic and a is an integer            from 1 to 15;        -   Z, which may or may not be present, is a peptide or            peptidomimetic having the formula (Xaa)_(b), wherein Xaa is            any amino acid or amino acid mimetic and b is an integer            from 1 to 15; and        -   A and B, at least one of which is present, are modifying            groups attached directly or indirectly to the amino terminus            and carboxy terminus, respectively, of the compound;    -   Xaa₁, Xaa₃, Y, Z, A and B being selected such that the compound        modulates the aggregation or inhibits the neurotoxicity of        natural β-amyloid peptides when contacted with the natural        β-amyloid peptides.

In this embodiment, the modulator compound is specifically modified ateither its amino-terminus, its carboxy-terminus, or both. Theterminology used in this formula is the same as described above.Suitable modifying groups are described in subsection II below. In oneembodiment, the compound is modified only at its amino terminus (i.e., Bis absent and the compound comprises the formula:A—(Y)—Xaa₁-Xaa₂-Xaa₃-Xaa₄—(Z)). In another embodiment, the compound ismodified only at its carboxy-terminus (i.e., A is absent and thecompound comprises the formula: (Y)—Xaa₁-Xaa₂-Xaa₃-Xaa₄—(Z)—B). In yetanother embodiment, the compound is modified at both its amino- andcarboxy termini (i.e., the compound comprises the formula:A—(Y)—Xaa₁-Xaa₂-Xaa₃-Xaa₄—(Z)—B and both A and B are present). Asdescribed above, the type and number of amino acid structures whichflank the Xaa₁-Xaa₂-Xaa₃-Xaa₄ core sequences in the above formula isvariable. For example, in one embodiment, a and b are integers from 1 to15. More preferably, a and b are integers between 1 and 10. Even morepreferably, a and b are integers between 1 and 5. Most preferably, a andb are integers between 1 and 3.

As demonstrated in Examples 7, 8 and 9, preferred Aβ modulator compoundsof the invention comprise modified forms of Aβ₁₄₋₂₁(His-Gln-Lys-Leu-Val-Phe-Phe-Ala; SEQ ID NO: 5), or amino-terminal orcarboxy-terminal deletions thereof, with a preferred “minimal coreregion” comprising Aβ₁₇₋₂₀. Accordingly, in specific embodiments, theinvention provides compounds comprising the formula:A—Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈—B

-   -   wherein        -   Xaa1 is a histidine structure;        -   Xaa2 is a glutamine structure;        -   Xaa3 is a lysine structure;        -   Xaa4 is a leucine structure;        -   Xaa5 is a valine structure;        -   Xaa6 is a phenylalanine structure;        -   Xaa7 is a phenylalanine structure;        -   Xaa8 is an alanine structure;        -   A and B are modifying groups attached directly or indirectly            to the amino terminus and carboxy terminus, respectively, of            the compound;    -   and wherein        -   Xaa₁-Xaa₂-Xaa₃, Xaa₁-Xaa₂ or Xaa₁ may or may not be present;        -   Xaa₈ may or may not be present; and        -   at least one of A and B is present.

In one specific embodiment, the compound comprises the formula:A—Xaa₄-Xaa₅-Xaa₆-Xaa₇—B (e.g, a modified form of Aβ₁₇₋₂₀, comprising anamino acid sequence Leu-Val-Phe-Phe; SEQ ID NO: 12).

In another specific embodiment, the compound comprises the formula:A—Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈—B (e.g, a modified form of Aβ₁₇₋₂₁,comprising an amino acid sequence Leu-Val-Phe-Phe-Ala; SEQ ID NO: 11).

In another specific embodiment, the compound comprises the formula:A—Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇—B (e.g., a modified form of Aβ₁₆₋₂₀,comprising an amino acid sequence Lys-Leu-Val-Phe-Phe; SEQ ID NO: 10).

In another specific embodiment, the compound comprises the formula:A—Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈—B (e.g., a modified form of Aβ₁₆₋₂₁,comprising an amino acid sequence Lys-Leu-Val-Phe-Phe-Ala; SEQ ID NO:9).

In another specific embodiment, the compound comprises the formula:A—Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇—B (e.g., a modified form of Aβ₁₅₋₂₀,comprising an amino acid sequence Gln-Lys-Leu-Val-Phe-Phe; SEQ ID NO:8).

In another specific embodiment, the compound comprises the formula:A—Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈—B (e.g., a modified form ofAβ₁₅₋₂₁, comprising an amino acid sequence Gln-Lys-Leu-Val-Phe-Phe-Ala;SEQ ID NO: 7).

In another specific embodiment, the compound comprises the formula:A—Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇—B (e.g., a modified form ofAβ₁₄₋₂₀, comprising an amino acid sequence His-Gln-Lys-Leu-Val-Phe-Phe;SEQ ID NO: 6).

In another specific embodiment, the compound comprises the formula:A—Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈—B (e.g., a modified form ofAβ₁₄₋₂₁, comprising an amino acid sequenceHis-Gln-Lys-Leu-Val-Phe-Phe-Ala; SEQ ID NO: 5).

In preferred embodiments of the aforementioned specific embodiments, Aor B is a cholanoyl structure or a biotin-containing structure(described further in subsection II below).

In further experiments to delineate subregions of Aβ upon which an Aβaggregation core domain can be modeled (the results of which aredescribed in Example 11), it was demonstrated that a modulator compoundhaving inhibitory activity can comprise as few as three Aβ amino acidsresidues (e.g., Val-Phe-Phe, which corresponds to Aβ₁₈₋₂₀ orPhe-Phe-Ala, which corresponds to Aβ₁₉₋₂₁). The results alsodemonstrated that a modulator compound having a modulating group at itscarboxy-terminus is effective at inhibiting Aβ aggregation. Stillfurther, the results demonstrated that the cholyl group, as a modulatinggroup, can be manipulated while maintaining the inhibitory activity ofthe compounds and that an iodotyrosyl can be substituted forphenylalanine (e.g., at position 19 or 20 of the Aβ sequence) whilemaintaining the ability of the compound to inhibit Aβ aggregation.

Still further, the results demonstrated that compounds with inhibitoryactivity can be created using amino acids residues that are derived fromthe Aβ sequence in the region of about positions 17-21 but wherein theamino acid sequence is rearranged or has a substitution with anon-Aβ-derived amino acid. Examples of such compounds include PPI-426,in which the sequence of Aβ₁₇₋₂₁ (LVFFA SEQ ID NO: 11) has beenrearranged (FFVLA SEQ ID NO: 21), PPI-372, in which the sequence ofAβ₁₆₋₂₀ (KLVFF SEQ ID NO: 10) has been rearranged (FKFVL SEQ ID NO: 29),and PPI-388, -389 and -390, in which the sequence of Aβ₁₇₋₂₁ (LVFFA SEQID NO: 11) has been substituted at position 17, 18 or 19, respectively,with an alanine residue (AVFFA (SEQ ID NO: 25) for PPI-388, LAFFA (SEQID NO: 13) for PPI-389 and LVAFA (SEQ ID NO: 33) for PPI-390). Theinhibitory activity of these compounds indicate that the presence in thecompound of an amino acid sequence directly corresponding to a portionof Aβ is not essential for inhibitory activity, but rather suggests thatmaintenance of the hydrophobic nature of this core region, by inclusionof amino acid residues such as phenylalanine, valine, leucine,regardless of their precise order, can be sufficient for inhibition ofAβ aggregation. Accordingly, an Aβ aggregation core domain can bedesigned based on the direct Aβ amino acid sequence or can be designedbased on a rearranged Aβ sequence which maintains the hydrophobicity ofthe Aβ subregion, e.g., the region around positions 17-20. This regionof Aβ contains the amino acid residues Leu, Val and Phe. Accordingly,preferred Aβ aggregation core domains are composed of at least threeamino acid structures (as that term is defined hereinbefore, includingamino acid derivatives, analogues and mimetics), wherein at least two ofthe amino acid structures are, independently, either a leucinestructure, a valine structure or a phenylalanine structure (as thoseterms are defined hereinbefore, including derivatives, analogues andmimetics).

Thus, in another embodiment, the invention provides a β-amyloidmodulator compound comprising a formula:

-   -   wherein        -   Xaa₁, Xaa₂ and Xaa₃ are each amino acid structures and at            least two of Xaa₁, Xaa₂ and Xaa₃ are, independently,            selected from the group consisting of a leucine structure, a            phenylalanine structure and a valine structure;        -   Y, which may or may not be present, is a peptidic structure            having the formula (Xaa)_(a), wherein Xaa is any amino acid            structure and a is an integer from 1 to 15;        -   Z, which may or may not be present, is a peptidic structure            having the formula (Xaa)_(b), wherein Xaa is any amino acid            structure and b is an integer from 1 to 15; and        -   A is a modifying group attached directly or indirectly to            the compound and n is an integer;    -   Xaa₁, Xaa₂, Xaa₃, Y, Z, A and n being selected such that the        compound modulates the aggregation or inhibits the neurotoxicity        of natural β-amyloid peptides when contacted with the natural        β-amyloid peptides.

Preferably, the compound inhibits aggregation of natural β-amyloidpeptides when contacted with the natural β-amyloid peptides. Inpreferred embodiments, Xaa₁ and Xaa₂ are each phenylalanine structuresor Xaa₂ and Xaa₃ are each phenylalanine structures. “n” can be, forexample, an integer between 1 and 5, whereas “a” and “b” can be, forexample, integers between 1 and 5. The modifying group “A” preferablycomprises a cyclic, heterocyclic or polycyclic group. More preferably, Acontains a cis-decalin group, such as cholanoyl structure or a cholylgroup In other embodiments, A can comprise a biotin-containing group, adiethylene-triaminepentaacetyl group, a (−)-menthoxyacetyl group, afluorescein-containing group or an N-acetylneuraminyl group. In yetother embodiments, the compound may promotes aggregation of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides,may be further modified to alter a pharmacokinetic property of thecompound or may be further modified to label the compound with adetectable substance.

In another embodiment, the invention provides a β-amyloid modulatorcompound comprising a formula:A—(Y)—Xaa₁-Xaa₂-Xaa₃—(Z)—B

-   -   wherein        -   Xaa₁, Xaa₂ and Xaa₃ are each amino acid structures and at            least two of Xaa₁, Xaa₂ and Xaa₃ are, independently,            selected from the group consisting of a leucine structure, a            phenylalanine structure and a valine structure;        -   Y, which may or may not be present, is a peptidic structure            having the formula (Xaa)_(a), wherein Xaa is any amino acid            structure and a is an integer from 1 to 15;        -   Z, which may or may not be present, is a peptidic structure            having the formula (Xaa)_(b), wherein Xaa is any amino acid            structure and b is an integer from 1 to 15; and        -   A and B, at least one of which is present, are modifying            groups attached directly or indirectly to the amino terminus            and carboxy terminus, respectively, of the compound;        -   Xaa₁, Xaa₂, Xaa₃, Y, Z, A and B being selected such that the            compound modulates the aggregation or inhibits the            neurotoxicity of natural β-amyloid peptides when contacted            with the natural β-amyloid peptides.

Preferably, the compound inhibits aggregation of natural β-amyloidpeptides when contacted with the natural β-amyloid peptides. Inpreferred embodiments, Xaa₁ and Xaa₂ are each phenylalanine structuresor Xaa₂ and Xaa₃ are each phenylalanine structures. In onesubembodiment, the compound comprises the formula:A—(Y)—Xaa₁-Xaa₂-Xaa₃—(Z)In another subembodiment, the compound comprises the formula:(Y)—Xaa₁-Xaa₂-Xaa₃—(Z)—B“n” can be, for example, an integer between 1 and 5, whereas “a” and “b”can be, for example, integers between 1 and 5. The modifying group “A”preferably comprises a cyclic, heterocyclic or polycyclic group. Morepreferably, A contains a cis-decalin group, such as cholanoyl structureor a cholyl group In other embodiments, A can comprise abiotin-containing group, a diethylene-triaminepentaacetyl group, a(−)-menthoxyacetyl group, a fluorescein-containing group or anN-acetylneuraminyl group. In yet other embodiments, the compound maypromote aggregation of natural β-amyloid peptides when contacted withthe natural β-amyloid peptides, may be further modified to alter apharmacokinetic property of the compound or may be further modified tolabel the compound with a detectable substance.

In preferred specific embodiments, the invention provides a β-amyloidmodulator compound comprising a modifying group attached directly orindirectly to a peptidic structure, wherein the peptidic structurecomprises amino acid structures having an amino acid sequence selectedfrom the group consisting of His-Gln-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO:5), His-Gln-Lys-Leu-Val-Phe-Phe (SEQ ID NO: 6),Gln-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: 7), Gln-Lys-Leu-Val-Phe-Phe (SEQID NO: 8), Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: 9), Lys-Leu-Val-Phe-Phe(SEQ ID NO: 10), Leu-Val-Phe-Phe-Ala (SEQ ID NO: 11), Leu-Val-Phe-Phe(SEQ ID NO: 12), Leu-Ala-Phe-Phe-Ala (SEQ ID NO: 13), Val-Phe-Phe (SEQID NO: 19), Phe-Phe-Ala (SEQ ID NO: 20), Phe-Phe-Val-Leu-Ala (SEQ ID NO:21), Leu-Val-Phe-Phe-Lys (SEQ ID NO: 22), Leu-Val-Iodotyrosine-Phe-Ala(SEQ ID NO: 23), Val-Phe-Phe-Ala (SEQ ID NO: 24), Ala-Val-Phe-Phe-Ala(SEQ ID NO: 25), Leu-Val-Phe-Iodotyrosine-Ala (SEQ ID NO: 26),Leu-Val-Phe-Phe-Ala-Glu (SEQ ID NO: 27), Phe-Phe-Val-Leu (SEQ ID NO:28), Phe-Lys-Phe-Val-Leu (SEQ ID NO: 29), Lys-Leu-Val-Ala-Phe (SEQ IDNO: 30), Lys-Leu-Val-Phe-Phe-βAla (SEQ ID NO: 31) andLeu-Val-Phe-Phe-DAla (SEQ ID NO: 32).

These specific compounds can be further modified to alter apharmacokinetic property of the compound and/or further modified tolabel the compound with a detectable substance.

The modulator compounds of the invention can be incorporated intopharmaceutical compositions (described further in subsection V below)and can be used in detection and treatment methods as described furtherin subsection VI below.

II. Modifying Groups

Within a modulator compound of the invention, a peptidic structure (suchas an Aβ derived peptide, or an Aβ aggregation core domain, or an aminoacid sequence corresponding to a rearranged Aβ aggregation core domain)is coupled directly or indirectly to at least one modifying group(abbreviated as MG). In one embodiment, a modulator compounds of theinvention comprising an aggregation core domain coupled to a modifyinggroup, the compound can be illustrated schematically as MG-ACD. The term“modifying group” is intended to include structures that are directlyattached to the peptidic structure (e.g., by covalent coupling), as wellas those that are indirectly attached to the peptidic structure (e.g.,by a stable non-covalent association or by covalent coupling toadditional amino acid residues, or mimetics, analogues or derivativesthereof, which may flank the Aβ-derived peptidic structure). Forexample, the modifying group can be coupled to the amino-terminus orcarboxy-terminus of an Aβ-derived peptidic structure, or to a peptidicor peptidomimetic region flanking the core domain. Alternatively, themodifying group can be coupled to a side chain of at least one aminoacid residue of an Aβ-derived peptidic structure, or to a peptidic orpeptidomimetic region flanking the core domain (e.g., through theepsilon amino group of a lysyl residue(s), through the carboxyl group ofan aspartic acid residue(s) or a glutamic acid residue(s), through ahydroxy group of a tyrosyl residue(s), a serine residue(s) or athreonine residue(s) or other suitable reactive group on an amino acidside chain). Modifying groups covalently coupled to the peptidicstructure can be attached by means and using methods well known in theart for linking chemical structures, including, for example, amide,alkylamino, carbamate or urea bonds.

The term “modifying group” is intended to include groups that are notnaturally coupled to natural Aβ peptides in their native form.Accordingly, the term “modifying group” is not intended to includehydrogen. The modifying group(s) is selected such that the modulatorcompound alters, and preferably inhibits, aggregation of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides orinhibits the neurotoxicity of natural β-amyloid peptides when contactedwith the natural β-amyloid peptides. Although not intending to belimited by mechanism, the modifying group(s) of the modulator compoundsof the invention is thought to function as a key pharmacophore which isimportant for conferring on the modulator the ability to disrupt Aβpolymerization.

In a preferred embodiment, the modifying group(s) comprises a cyclic,heterocyclic or polycyclic group. The term “cyclic group”, as usedherein, is intended to include cyclic saturated or unsaturated (i.e.,aromatic) group having from about 3 to 10, preferably about 4 to 8, andmore preferably about 5 to 7, carbon atoms. Exemplary cyclic groupsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, andcyclooctyl. Cyclic groups may be unsubstituted or substituted at one ormore ring positions. Thus, a cyclic group may be substituted with, e.g.,halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, heterocycles,hydroxyls, aminos, nitros, thiols amines, imines, amides, phosphonates,phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls,sulfonates, selenoethers, ketones, aldehydes, esters, —CF₃, —CN, or thelike.

The term “heterocyclic group” is intended to include cyclic saturated orunsaturated (i.e., aromatic) group having from about 3 to 10, preferablyabout 4 to 8, and more preferably about 5 to 7, carbon atoms, whereinthe ring structure includes about one to four heteroatoms. Heterocyclicgroups include pyrrolidine, oxolane, thiolane, imidazole, oxazole,piperidine, piperazine, morpholine. The heterocyclic ring can besubstituted at one or more positions with such substituents as, forexample, halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, otherheterocycles, hydroxyl, amino, nitro, thiol, amines, imines, amides,phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers,thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, —CF₃,—CN, or the like. Heterocycles may also be bridged or fused to othercyclic groups as described below.

The term “polycyclic group” as used herein is intended to refer to twoor more saturated or unsaturated (i.e., aromatic) cyclic rings in whichtwo or more carbons are common to two adjoining rings, e.g., the ringsare “fused rings”. Rings that are joined through non-adjacent atoms aretermed “bridged” rings. Each of the rings of the polycyclic group can besubstituted with such substituents as described above, as for example,halogens, alkyls, cycloalkyls, alkenyls, alkynyls, hydroxyl, amino,nitro, thiol, amines, imines, amides, phosphonates, phosphines,carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls,selenoethers, ketones, aldehydes, esters, —CF₃, —CN, or the like.

A preferred polycyclic group is a group containing a cis-decalinstructure. Although not intending to be limited by mechanism, it isthought that the “bent” conformation conferred on a modifying group bythe presence of a cis-decalin structure contributes to the efficacy ofthe modifying group in disrupting Aβ polymerization. Accordingly, otherstructures which mimic the “bent” configuration of the cis-decalinstructure can also be used as modifying groups. An example of acis-decalin containing structure that can be used as a modifying groupis a cholanoyl structure, such as a cholyl group. For example, amodulator compound can be modified at its amino terminus with a cholylgroup by reacting the aggregation core domain with cholic acid, a bileacid, as described in Example 4 (the structure of cholic acid isillustrated in FIG. 2). Moreover, a modulator compound can be modifiedat its carboxy terminus with a cholyl group according to methods knownin the art (see e.g., Wess, G. et al. (1993) Tetrahedron Letters,34:817-822; Wess, G. et al. (1992) Tetrahedron Letters 33:195-198; andKramer, W. et al. (1992) J. Biol. Chem. 267:18598-18604). Cholylderivatives and analogues can also be used as modifying groups. Forexample, a preferred cholyl derivative is Aic(3-(O-aminoethyl-iso)-cholyl), which has a free amino group that can beused to further modify the modulator compound (e.g., a chelation groupfor ^(99m)Tc can be introduced through the free amino group of Aic). Asused herein, the term “cholanoyl structure” is intended to include thecholyl group and derivatives and analogues thereof, in particular thosewhich retain a four-ring cis-decalin configuration. Examples ofcholanoyl structures include groups derived from other bile acids, suchas deoxycholic acid, lithocholic acid, ursodeoxycholic acid,chenodeoxycholic acid and hyodeoxycholic acid, as well as other relatedstructures such as cholanic acid, bufalin and resibufogenin (althoughthe latter two compounds are not preferred for use as a modifyinggroup). Another example of a cis-decalin containing compound is5β-cholestan-3α-ol (the cis-decalin isomer of (+)-dihydrocholesterol).For further description of bile acid and steroid structure andnomenclature, see Nes, W. R. and McKean, M. L. Biochemistry of Steroidsand Other Isopentanoids, University Park Press, Baltimore, Md., Chapter2.

In addition to cis-decalin containing groups, other polycyclic groupsmay be used as modifying groups. For example, modifying groups derivedfrom steroids or β-lactams may be suitable modifying groups. Moreover,non-limiting examples of some additional cyclic, heterocyclic orpolycyclic compounds which can be used to modify an Aβ-derived peptidicstructure are shown schematically in FIG. 2. In one embodiment, themodifying group is a “biotinyl structure”, which includes biotinylgroups and analogues and derivatives thereof (such as a 2-iminobiotinylgroup). In another embodiment, the modifying group can comprise a“fluorescein-containing group”, such as a group derived from reacting anAβ-derived peptidic structure with 5-(and 6-)-carboxyfluorescein,succinimidyl ester or fluorescein isothiocyanate. In various otherembodiments, the modifying group(s) can comprise an N-acetylneuraminylgroup, a trans-4-cotininecarboxyl group, a 2-imino-1-imidazolidineacetylgroup, an (S)-(−)-indoline-2-carboxyl group, a (−)-menthoxyacetyl group,a 2-norbornaneacetyl group, a γ-oxo-5-acenaphthenebutyryl, a(−)-2-oxo-4-thiazolidinecarboxyl group, a tetrahydro-3-furoyl group, a2-iminobiotinyl group, a diethylenetriaminepentaacetyl group, a4-morpholinecarbonyl group, a 2-thiopheneacetyl group or a2-thiophenesulfonyl group.

Preferred modifying groups include groups comprising cholyl structures,biotinyl structures, fluorescein-containing groups, adiethylene-triaminepentaacetyl group, a (−)-menthoxyacetyl group, and aN-acetylneuraminyl group. More preferred modifying groups thosecomprising a cholyl structure or an iminiobiotinyl group.

In addition to the cyclic, heterocyclic and polycyclic groups discussedabove, other types of modifying groups can be used in a modulator of theinvention. For example, small hydrophobic groups may be suitablemodifying groups. An example of a suitable non-cyclic modifying group isan acetyl group.

Yet another type of modifying group is a compound that contains anon-natural amino acid that acts as a beta-turn mimetic, such as adibenzofuran-based amino acid described in Tsang, K. Y. et al. (1994) J.Am. Chem. Soc. 116:3988-4005; Diaz, H and Kelly, J. W. (1991)Tetrahedron Letters 41:5725-5728; and Diaz, H. et al. (1992) J. Am.Chem. Soc. 114:8316-8318. An example of such a modifying group is apeptide-aminoethyldibenzofuranyl-proprionic acid (Adp) group (e.g.,DDIIL-Adp (SEQ ID NO: 34)). This type of modifying group further cancomprise one or more N-methyl peptide bonds to introduce additionalsteric hindrance to the aggregation of natural β-AP when compounds ofthis type interact with natural β-AP.

III. Additional Chemical Modifications of Aβ Modulators

A β-amyloid modulator compound of the invention can be further modifiedto alter the specific properties of the compound while retaining theability of the compound to alter Aβ aggregation and inhibit Aβneurotoxicity. For example, in one embodiment, the compound is furthermodified to alter a pharmacokinetic property of the compound, such as invivo stability or half-life. In another embodiment, the compound isfurther modified to label the compound with a detectable substance. Inyet another embodiment, the compound is further modified to couple thecompound to an additional therapeutic moiety. Schematically, a modulatorof the invention comprising an Aβ aggregation core domain coupleddirectly or indirectly to at least one modifying group can beillustrated as MG-ACD, whereas this compound which has been furthermodified to alter the properties of the modulator can be illustrated asMG-ACD-CM, wherein CM represents an additional chemical modification.

To further chemically modify the compound, such as to alter thepharmacokinetic properties of the compound, reactive groups can bederivatized. For example, when the modifying group is attached to theamino-terminal end of the aggregation core domain, the carboxy-terminalend of the compound can be further modified. Preferred C-terminalmodifications include those which reduce the ability of the compound toact as a substrate for carboxypeptidases. Examples of preferredC-terminal modifiers include an amide group, an ethylamide group andvarious non-natural amino acids, such as D-amino acids and β-alanine.Alternatively, when the modifying group is attached to thecarboxy-terminal end of the aggregation core domain, the amino-terminalend of the compound can be further modified, for example, to reduce theability of the compound to act as a substrate for aminopeptidases.

A modulator compound can be further modified to label the compound byreacting the compound with a detectable substance. Suitable detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ¹⁴C, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I,^(99m)Tc, ³⁵S or ³H. In a preferred embodiment, a modulator compound isradioactively labeled with ¹⁴C, either by incorporation of ¹⁴C into themodifying group or one or more amino acid structures in the modulatorcompound. Labeled modulator compounds can be used to assess the in vivopharmacokinetics of the compounds, as well as to detect Aβ aggregation,for example for diagnostic purposes. Aβ aggregation can be detectedusing a labeled modulator compound either in vivo or in an in vitrosample derived from a subject.

Preferably, for use as an in vivo diagnostic agent, a modulator compoundof the invention is labeled with radioactive technetium or iodine.Accordingly, in one embodiment, the invention provides a modulatorcompound labeled with technetium, preferably ^(99m)Tc. Methods forlabeling peptide compounds with technetium are known in the art (seee.g., U.S. Pat. Nos. 5,443,815, 5,225,180 and 5,405,597, all by Dean etal.; Stepniak-Biniakiewicz, D., et al. (1992) J. Med. Chem. 35:274-279;Fritzberg, A. R., et al. (1988) Proc. Natl. Acad. Sci. USA 85:4025-4029;Baidoo, K. E., et al. (1990) Cancer Res. Suppl. 50:799s-803s; and Regan,L. and Smith, C. K. (1995) Science 270:980-982). A modifying group canbe chosen that provides a site at which a chelation group for ^(99m)Tccan be introduced, such as the Aic derivative of cholic acid, which hasa free amino group (see Example 11). In another embodiment, theinvention provides a modulator compound labeled with radioactive iodine.For example, a phenylalanine residue within the Aβ sequence (such asPhe₁₉ or Phe₂₀) can be substituted with radioactive iodotyrosyl (seeExample 11). Any of the various isotopes of radioactive iodine can beincorporated to create a diagnostic agent. Preferably, ¹²³I(half-life=13.2 hours) is used for whole body scintigraphy, ¹²⁴I (halflife=4 days) is used for positron emission tomography (PET), ¹²⁵I (halflife=60 days) is used for metabolic turnover studies and ¹³¹I (halflife=8 days) is used for whole body counting and delayed low resolutionimaging studies.

Furthermore, an additional modification of a modulator compound of theinvention can serve to confer an additional therapeutic property on thecompound. That is, the additional chemical modification can comprise anadditional functional moiety. For example, a functional moiety whichserves to break down or dissolve amyloid plaques can be coupled to themodulator compound. In this form, the MG-ACD portion of the modulatorserves to target the compound to Aβ peptides and disrupt thepolymerization of the Aβ peptides, whereas the additional functionalmoiety serves to break down or dissolve amyloid plaques after thecompound has been targeted to these sites.

In an alternative chemical modification, a β-amyloid compound of theinvention is prepared in a “prodrug” form, wherein the compound itselfdoes not modulate Aβ aggregation, but rather is capable of beingtransformed, upon metabolism in vivo, into a β-amyloid modulatorcompound as defined herein. For example, in this type of compound, themodulating group can be present in a prodrug form that is capable ofbeing converted upon metabolism into the form of an active modulatinggroup. Such a prodrug form of a modifying group is referred to herein asa “secondary modifying group.” A variety of strategies are known in theart for preparing peptide prodrugs that limit metabolism in order tooptimize delivery of the active form of the peptide-based drug (seee.g., Moss, J. (1995) in Peptide-Based Drug Design: ControllingTransport and Metabolism, Taylor, M. D. and Amidon, G. L. (eds), Chapter18. Additionally strategies have been specifically tailored to achievingCNS delivery based on “sequential metabolism” (see e.g., Bodor, N., etal. (1992) Science 257:1698-1700; Prokai, L., et al. (1994) J. Am. Chem.Soc. 116:2643-2644; Bodor, N. and Prokai, L. (1995) in Peptide-BasedDrug Design: Controlling Transport and Metabolism, Taylor, M. D. andAmidon, G. L. (eds), Chapter 14. In one embodiment of a prodrug form ofa modulator of the invention, the modifying group comprises an alkylester to facilitate blood-brain barrier permeability.

Modulator compounds of the invention can be prepared by standardtechniques known in the art. The peptide component of a modulatorcomposed, at least in part, of a peptide, can be synthesized usingstandard techniques such as those described in Bodansky, M. Principlesof Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant, G. A(ed.). Synthetic Peptides: A User's Guide, W.H. Freeman and Company, NewYork (1992). Automated peptide synthesizers are commercially available(e.g., Advanced ChemTech Model 396; Milligen/Biosearch 9600).Additionally, one or more modulating groups can be attached to theAβ-derived peptidic component (e.g., an Aβ aggregation core domain) bystandard methods, for example using methods for reaction through anamino group (e.g., the alpha-amino group at the amino-terminus of apeptide), a carboxyl group (e.g., at the carboxy terminus of a peptide),a hydroxyl group (e.g., on a tyrosine, seine or threonine residue) orother suitable reactive group on an amino acid side chain (see e.g.,Greene, T. W and Wuts, P. G. M. Protective Groups in Organic Synthesis,John Wiley and Sons, Inc., New York (1991)). Exemplary syntheses ofpreferred β amyloid modulators is described further in Examples 1, 4 and11.

IV. Screening Assays

Another aspect of the invention pertains to a method for selecting amodulator of β-amyloidaggregation. In the method, a test compound iscontacted with natural β amyloid peptides, the aggregation of thenatural β-AP is measured and a modulator is selected based on theability of the test compound to alter the aggregation of the naturalβ-AP (e.g., inhibit or promote aggregation). In a preferred embodiment,the test compound is contacted with a molar excess amount of the naturalβ-AP. The amount and/or rate of natural β-AP aggregation in the presenceof the test compound can be determined by a suitable assay indicative ofβ-AP aggregation, as described herein (see e.g., Examples 2, 5 and 6).

In a preferred assay, the natural β-AP is dissolved in solution in thepresence of the test compound and aggregation of the natural β-AP isassessed in a nucleation assay (see Example 6) by assessing theturbidity of the solution over time, as measured by the apparentabsorbance of the solution at 405 nm (described further in Example 6;see also Jarrett et al. (1993) Biochemistry 32:4693-4697). In theabsence of a β-amyloid modulator, the A_(405 nm) of the solutiontypically stays relatively constant during a lag time in which the β-APremains in solution, but then the A_(405 nm) of the solution rapidlyincreases as the β-AP aggregates and comes out of solution, ultimatelyreaching a plateau level (i.e., the A_(405 nm) of the solution exhibitssigmoidal kinetics over time). In contrast, in the presence of a testcompound that inhibits β-AP aggregation, the A_(405 nm) of the solutionis reduced compared to when the modulator is absent. Thus, in thepresence of the inhibitory modulator, the solution may exhibit anincreased lag time, a decreased slope of aggregation and/or a lowerplateau level compared to when the modulator is absent. This method forselecting a modulator of β-amyloid polymerization can similarly be usedto select modulators that promote β-AP aggregation. Thus, in thepresence of a modulator that promotes β-AP aggregation, the A_(405 nm)of the solution is increased compared to when the modulator is absent(e.g., the solution may exhibit an decreased lag time, increase slope ofaggregation and/or a higher plateau level compared to when the modulatoris absent).

Another assay suitable for use in the screening method of the invention,a seeded extension assay, is also described further in Example 6. Inthis assay, β-AP monomer and an aggregated β-AP “seed” are combined, inthe presence and absence of a test compound, and the amount of β-fibrilformation is assayed based on enhanced emission of the dye Thioflavine Twhen contacted with β-AP fibrils. Moreover, β-AP aggregation can beassessed by electron microscopy (EM) of the β-AP preparation in thepresence or absence of the modulator. For example, β amyloid fibrilformation, which is detectable by EM, is reduced in the presence of amodulator that inhibits β-AP aggregation (i.e., there is a reducedamount or number of β-fibrils in the presence of the modulator), whereasβ fibril formation is increased in the presence of a modulator thatpromotes β-AP aggregation (i.e., there is an increased amount or numberof β-fibrils in the presence of the modulator).

An even more preferred assay for use in the screening method of theinvention to select suitable modulators is the neurotoxicity assaydescribed in Examples 3 and 10. Compounds are selected which inhibit theformation of neurotoxic Aβ aggregates and/or which inhibit theneurotoxicity of preformed Aβ fibrils. This neurotoxicity assay isconsidered to be predictive of neurotoxicity in vivo. Accordingly,inhibitory activity of a modulator compound in the in vitroneurotoxicity assay is predictive of similar inhibitory activity of thecompound for neurotoxicity in vivo.

V. Pharmaceutical Compositions

Another aspect of the invention pertains to pharmaceutical compositionsof the β-amyloid modulator compounds of the invention. In oneembodiment, the composition includes a β amyloid modulator compound in atherapeutically or prophylactically effective amount sufficient toalter, and preferably inhibit, aggregation of natural β-amyloidpeptides, and a pharmaceutically acceptable carrier. In anotherembodiment, the composition includes a β amyloid modulator compound in atherapeutically or prophylactically effective amount sufficient toinhibit the neurotoxicity of natural β-amyloid peptides, and apharmaceutically acceptable carrier. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired therapeutic result, such asreduction or reversal or β-amyloid deposition and/or reduction orreversal of Aβ neurotoxicity. A therapeutically effective amount ofmodulator may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of the modulator toelicit a desired response in the individual. Dosage regimens may beadjusted to provide the optimum therapeutic response. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the modulator are outweighed by the therapeutically beneficialeffects. The potential neurotoxicity of the modulators of the inventioncan be assayed using the cell-based assay described in Examples 3 and 10and a therapeutically effective modulator can be selected which does notexhibit significant neurotoxicity. In a preferred embodiment, atherapeutically effective amount of a modulator is sufficient to alter,and preferably inhibit, aggregation of a molar excess amount of naturalβ-amyloid peptides. A “prophylactically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired prophylactic result, such as preventing orinhibiting the rate of β-amyloid deposition and/or Aβ neurotoxicity in asubject predisposed to β-amyloid deposition. A prophylacticallyeffective amount can be determined as described above for thetherapeutically effective amount. Typically, since a prophylactic doseis used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

One factor that may be considered when determining a therapeutically orprophylactically effective amount of a β amyloid modulator is theconcentration of natural β-AP in a biological compartment of a subject,such as in the cerebrospinal fluid (CSF) of the subject. Theconcentration of natural β-AP in the CSF has been estimated at 3 nM(Schwartzman, (1994) Proc. Natl. Acad. Sci. USA 91:8368-8372). Anon-limiting range for a therapeutically or prophylactically effectiveamounts of a β amyloid modulator is 0.01 nM-10 μM. It is to be notedthat dosage values may vary with the severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

The amount of active compound in the composition may vary according tofactors such as the disease state, age, sex, and weight of theindividual, each of which may affect the amount of natural β-AP in theindividual. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

As used herein “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. In one embodiment, the carrier is suitablefor parenteral administration. Preferably, the carrier is suitable foradministration into the central nervous system (e.g., intraspinally orintracerebrally). Alternatively, the carrier can be suitable forintravenous, intraperitoneal or intramuscular administration. In anotherembodiment, the carrier is suitable for oral administration.Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, monostearate salts and gelatin. Moreover, the modulators can beadministered in a time release formulation, for example in a compositionwhich includes a slow release polymer. The active compounds can beprepared with carriers that will protect the compound against rapidrelease, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, polylactic acid andpolylactic, polyglycolic copolymers (PLG). Many methods for thepreparation of such formulations are patented or generally known tothose skilled in the art.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., β-amyloid modulator) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

A modulator compound of the invention can be formulated with one or moreadditional compounds that enhance the solubility of the modulatorcompound. Preferred compounds to be added to formulations to enhance thesolubility of the modulators are cyclodextrin derivatives, preferablyhydroxypropyl-γ-cyclodextrin. Drug delivery vehicles containing acyclodextrin derivative for delivery of peptides to the central nervoussystem are described in Bodor, N., et al. (1992) Science 257:1698-1700.For the β-amyloid modulators described herein, inclusion in theformulation of hydroxypropyl-γ-cyclodextrin at a concentration 50-200 mMincreases the aqueous solubility of the compounds. In addition toincreased solubility, inclusion of a cyclodextrin derivative in theformulation may have other beneficial effects, since β-cyclodextrinitself has been reported to interact with the Aβ peptide and inhibitfibril formation in vitro (Camilleri, P., et al (1994) FEBS Letters341:256-258. Accordingly, use of a modulator compound of the inventionin combination with a cyclodextrin derivative may result in greaterinhibition of Aβ aggregation than use of the modulator alone. Chemicalmodifications of cyclodextrins are known in the art (Hanessian, S., etal. (1995) J. Org. Chem. 60:4786-4797). In addition to use as anadditive in a pharmaceutical composition containing a modulator of theinvention, cyclodextrin derivatives may also be useful as modifyinggroups and, accordingly, may also be covalently coupled to an Aβ peptidecompound to form a modulator compound of the invention.

In another embodiment, a pharmaceutical composition comprising amodulator of the invention is formulated such that the modulator istransported across the blood-brain barrier (BBB). Various strategiesknown in the art for increasing transport across the BBB can be adaptedto the modulators of the invention to thereby enhance transport of themodulators across the BBB (for reviews of such strategies, see e.g.,Pardridge, W. M. (1994) Trends in Biotechnol. 12:239-245; Van Bree, J.B. et al. (1993) Pharm. World Sci. 15:2-9; and Pardridge, W. M. et al.(1992) Pharmacol. Toxicol. 71:3-10). In one approach, the modulator ischemically modified to form a prodrug with enhanced transmembranetransport. Suitable chemical modifications include covalent linking of afatty acid to the modulator through an amide or ester linkage (see e.g.,U.S. Pat. No. 4,933,324 and PCT Publication WO 89/07938, both byShashoua; U.S. Pat. No. 5,284,876 by Hesse et al.; Toth, I. et al.(1994) J. Drug Target. 2:217-239; and Shashoua, V. E. et al. (1984) J.Med. Chem. 27:659-664) and glycating the modulator (see e.g., U.S. Pat.No. 5,260,308 by Poduslo et al.). Also, N-acylamino acid derivatives maybe used in a modulator to form a “lipidic” prodrug (see e.g., U.S. Pat.No. 5,112,863 by Hashimoto et al).

In another approach for enhancing transport across the BBB, a peptidicor peptidomimetic modulator is conjugated to a second peptide orprotein, thereby forming a chimeric protein, wherein the second peptideor protein undergoes absorptive-mediated or receptor-mediatedtranscytosis through the BBB. Accordingly, by coupling the modulator tothis second peptide or protein, the chimeric protein is transportedacross the BBB. The second peptide or protein can be a ligand for abrain capillary endothelial cell receptor ligand. For example, apreferred ligand is a monoclonal antibody that specifically binds to thetransferrin receptor on brain capillary endothelial cells (see e.g.,U.S. Pat. Nos. 5,182,107 and 5,154,924 and PCT Publications WO 93/10819and WO 95/02421, all by Friden et al.). Other suitable peptides orproteins that can mediate transport across the BBB include histones (seee.g., U.S. Pat. No. 4,902,505 by Pardridge and Schimmel) and ligandssuch as biotin, folate, niacin, pantothenic acid, riboflavin, thiamin,pryridoxal and ascorbic acid (see e.g., U.S. Pat. Nos. 5,416,016 and5,108,921, both by Heinstein). Additionally, the glucose transporterGLUT-1 has been reported to transport glycopeptides(L-serinyl-β-D-glucoside analogues of [Met5]enkephalin) across the BBB(Polt, R. et al. (1994) Proc. Natl. Acad. Sci. USA 91:7114-1778).Accordingly, a modulator compound can be coupled to such a glycopeptideto target the modulator to the GLUT-1 glucose transporter. For example,a modulator compound which is modified at its amino terminus with themodifying group Aic (3-(O-aminoethyl-iso)-cholyl, a derivative of cholicacid having a free amino group) can be coupled to a glycopeptide throughthe amino group of Aic by standard methods. Chimeric proteins can beformed by recombinant DNA methods (e.g., by formation of a chimeric geneencoding a fusion protein) or by chemical crosslinking of the modulatorto the second peptide or protein to form a chimeric protein. Numerouschemical crosslinking agents are known in the (e.g., commerciallyavailable from Pierce, Rockford Ill.). A crosslinking agent can bechosen which allows for high yield coupling of the modulator to thesecond peptide or protein and for subsequent cleavage of the linker torelease bioactive modulator. For example, a biotin-avidin-based linkersystem may be used.

In yet another approach for enhancing transport across the BBB, themodulator is encapsulated in a carrier vector which mediates transportacross the BBB. For example, the modulator can be encapsulated in aliposome, such as a positively charged unilamellar liposome (see e.g.,PCT Publications WO 88/07851 and WO 88/07852, both by Faden) or inpolymeric microspheres (see e.g., U.S. Pat. No. 5,413,797 by Khan etal., U.S. Pat. No. 5,271,961 by Mathiowitz et al. and U.S. Pat. No.5,019,400 by Gombotz et al.). Moreover, the carrier vector can bemodified to target it for transport across the BBB. For example, thecarrier vector (e.g., liposome) can be covalently modified with amolecule which is actively transported across the BBB or with a ligandfor brain endothelial cell receptors, such as a monoclonal antibody thatspecifically binds to transferrin receptors (see e.g., PCT PublicationsWO 91/04014 by Collins et al and WO 94/02178 by Greig et al.).

In still another approach to enhancing transport of the modulator acrossthe BBB, the modulator is coadministered with another agent whichfunctions to permeabilize the BBB. Examples of such BBB “permeabilizers”include bradykinin and bradykinin agonists (see e.g., U.S. Pat. No.5,112,596 by Malfroy-Camine) and peptidic compounds disclosed in U.S.Pat. No. 5,268,164 by Kozarich et al.

A modulator compound of the invention can be formulated into apharmaceutical composition wherein the modulator is the only activecompound or, alternatively, the pharmaceutical composition can containadditional active compounds. For example, two or more modulatorcompounds may be used in combination. Moreover, a modulator compound ofthe invention can be combined with one or more other agents that haveanti-amyloidogenic properties. For example, a modulator compound can becombined with the non-specific cholinesterase inhibitor tacrine(COGNEX®, Parke-Davis).

In another embodiment, a pharmaceutical composition of the invention isprovided as a packaged formulation. The packaged formulation may includea pharmaceutical composition of the invention in a container and printedinstructions for administration of the composition for treating asubject having a disorder associated with β-amyloidosis, e.g.Alzheimer's disease.

VI. Methods of Using Aβ Modulators

Another aspect of the invention pertains to methods for altering theaggregation or inhibiting the neurotoxicity of natural β-amyloidpeptides. In the methods of the invention, natural β amyloid peptidesare contacted with a β amyloid modulator such that the aggregation ofthe natural β amyloid peptides is altered or the neurotoxicity of thenatural β amyloid peptides is inhibited. In a preferred embodiment, themodulator inhibits aggregation of the natural β amyloid peptides. Inanother embodiment, the modulator promotes aggregation of the natural βamyloid peptides. Preferably, aggregation of a molar excess amount ofβ-AP, relative to the amount of modulator, is altered upon contact withthe modulator.

In the method of the invention, natural β amyloid peptides can becontacted with a modulator either in vitro or in vivo. Thus, the term“contacted with” is intended to encompass both incubation of a modulatorwith a natural β-AP preparation in vitro and delivery of the modulatorto a site in vivo where natural β-AP is present. Since the modulatorcompound interacts with natural β-AP, the modulator compounds can beused to detect natural β-AP, either in vitro or in vivo. Accordingly,one use of the modulator compounds of the invention is as diagnosticagents to detect the presence of natural β-AP, either in a biologicalsample or in vivo in a subject. Furthermore, detection of natural β-APutilizing a modulator compound of the invention further can be used todiagnose amyloidosis in a subject. Additionally, since the modulatorcompounds of the invention disrupt β-AP aggregation and inhibit β-APneurotoxicity, the modulator compounds also are useful in the treatmentof disorders associated with β-amyloidosis, either prophylactically ortherapeutically. Accordingly, another use of the modulator compounds ofthe invention is as therapeutic agents to alter aggregation and/orneurotoxicity of natural β-AP.

In one embodiment, a modulator compound of the invention is used invitro, for example to detect and quantitate natural β-AP in sample(e.g., a sample of biological fluid). To aid in detection, the modulatorcompound can be modified with a detectable substance. The source ofnatural β-AP used in the method can be, for example, a sample ofcerebrospinal fluid (e.g., from an AD patient, an adult susceptible toAD due to family history, or a normal adult). The natural β-AP sample iscontacted with a modulator of the invention and aggregation of the β-APis measured, such as by as assay described in Examples 2, 5 and 6.Preferably, the nucleation assay and/or seeded extension assay describedin Example 6 is used. The degree of aggregation of the S-AP sample canthen be compared to that of a control sample(s) of a known concentrationof β-AP, similarly contacted with the modulator and the results can beused as an indication of whether a subject is susceptible to or has adisorder associated with β-amyloidosis. Moreover, β-AP can be detectedby detecting a modulating group incorporated into the modulator. Forexample, modulators incorporating a biotin compound as described herein(e.g., an amino-terminally biotinylated β-AP peptide) can be detectedusing a streptavidin or avidin probe which is labeled with a detectablesubstance (e.g., an enzyme, such as peroxidase). Detection of naturalβ-AP aggregates mixed with a modulator of the invention using a probethat binds to the modulating group (e.g., biotin/streptavidin) isdescribed further in Example 2.

In another embodiment, a modulator compound of the invention is used invivo to detect, and, if desired, quantitate, natural β-AP deposition ina subject, for example to aid in the diagnosis of β amyloidosis in thesubject. To aid in detection, the modulator compound can be modifiedwith a detectable substance, preferably ^(99m)Tc or radioactive iodine(described further above), which can be detected in vivo in a subject.The labeled β-amyloid modulator compound is administered to the subjectand, after sufficient time to allow accumulation of the modulator atsites of amyloid deposition, the labeled modulator compound is detectedby standard imaging techniques. The radioactive signal generated by thelabeled compound can be directly detected (e.g., whole body counting),or alternatively, the radioactive signal can be converted into an imageon an autoradiograph or on a computer screen to allow for imaging ofamyloid deposits in the subject. Methods for imaging amyloidosis usingradiolabeled proteins are known in the art. For example, serum amyloid Pcomponent (SAP), radiolabeled with either ¹²³I or ^(99m)Tc, has beenused to image systemic amyloidosis (see e.g., Hawkins, P. N. and Pepys,M. B. (1995) Eur. J. Nucl. Med. 22:595-599). Of the various isotypes ofradioactive iodine, preferably ¹²³I (half-life=13.2 hours) is used forwhole body scintigraphy, ¹²⁴I (half life=4 days) is used for positronemission tomography (PET), ¹²⁵I (half life=60 days) is used formetabolic turnover studies and ¹³¹I (half life=8 days) is used for wholebody counting and delayed low resolution imaging studies. Analogous tostudies using radiolabeled SAP, a labeled modulator compound of theinvention can be delivered to a subject by an appropriate route (e.g.,intravenously, intraspinally, intracerebrally) in a single bolus, forexample containing 100 μg of labeled compound carrying approximately 180MBq of radioactivity.

The invention provides a method for detecting the presence or absence ofnatural β-amyloid peptides in a biological sample, comprising contactinga biological sample with a compound of the invention and detecting thecompound bound to natural β-amyloid peptides to thereby detect thepresence or absence of natural β-amyloid peptides in the biologicalsample. In one embodiment, the β-amyloid modulator compound and thebiological sample are contacted in vitro. In another embodiment, theβ-amyloid modulator compound is contacted with the biological sample byadministering the β-amyloid modulator compound to a subject. For in vivoadministration, preferably the compound is labeled with radioactivetechnetium or radioactive iodine.

The invention also provides a method for detecting natural β-amyloidpeptides to facilitate diagnosis of a β-amyloidogenic disease,comprising contacting a biological sample with the compound of theinvention and detecting the compound bound to natural β-amyloid peptidesto facilitate diagnosis of a β-amyloidogenic disease. In one embodiment,the β-amyloid modulator compound and the biological sample are contactedin vitro. In another embodiment, the β-amyloid modulator compound iscontacted with the biological sample by administering the β-amyloidmodulator compound to a subject. For in vivo administration, preferablythe compound is labeled with radioactive technetium or radioactiveiodine. Preferably, use of the method facilitates diagnosis ofAlzheimer's disease.

In another embodiment, the invention provides a method for alteringnatural β-AP aggregation or inhibiting β-AP neurotoxicity, which can beused prophylactically or therapeutically in the treatment or preventionof disorders associated with β amyloidosis, e.g., Alzheimer's Disease.As demonstrated in Example 10, modulator compounds of the inventionreduce the toxicity of natural β-AP aggregates to cultured neuronalcells. Moreover, the modulators not only reduce the formation ofneurotoxic aggregates but also have the ability to reduce theneurotoxicity of preformed Aβ fibrils. Accordingly, the modulatorcompounds of the invention can be used to inhibit or prevent theformation of neurotoxic Aβ fibrils in subjects (e.g., prophylacticallyin a subject predisposed to β-amyloid deposition) and can be used toreverse β-amyloidosis therapeutically in subjects already exhibitingβ-amyloid deposition.

A modulator of the invention is contacted with natural β amyloidpeptides present in a subject (e.g., in the cerebrospinal fluid orcerebrum of the subject) to thereby alter the aggregation of the naturalβ-AP and/or inhibit the neurotoxicity of the natural β-APs. A modulatorcompound alone can be administered to the subject, or alternatively, themodulator compound can be administered in combination with othertherapeutically active agents (e.g., as discussed above in subsectionIV). When combination therapy is employed, the therapeutic agents can becoadministered in a single pharmaceutical composition, coadministered inseparate pharmaceutical compositions or administered sequentially.

The modulator may be administered to a subject by any suitable routeeffective for inhibiting natural β-AP aggregation in the subject,although in a particularly preferred embodiment, the modulator isadministered parenterally, most preferably to the central nervous systemof the subject. Possible routes of CNS administration includeintraspinal administration and intracerebral administration (e.g.,intracerebrovascular administration). Alternatively, the compound can beadministered, for example, orally, intraperitoneally, intravenously orintramuscularly. For non-CNS administration routes, the compound can beadministered in a formulation which allows for transport across the BBB.Certain modulators may be transported across the BBB without anyadditional further modification whereas others may need furthermodification as described above in subsection IV.

Suitable modes and devices for delivery of therapeutic compounds to theCNS of a subject are known in the art, including cerebrovascularreservoirs (e.g., Ommaya or Rikker reservoirs; see e.g., Raney, J. P. etal. (1988) J. Neurosci. Nurs. 20:23-29; Sundaresan, N. et al. (1989)Oncology 3:15-22), catheters for intrathecal delivery (e.g.,Port-a-Cath, Y-catheters and the like; see e.g., Plummer, J. L. (1991)Pain 44:215-220; Yaksh, T. L. et al. (1986) Pharmacol. Biochem. Behav.25:483-485), injectable intrathecal reservoirs (e.g., Spinalgesic; seee.g., Brazenor, G. A. (1987) Neurosurgery 21:484-491), implantableinfusion pump systems (e.g., Infusaid; see e.g., Zierski, J. et al.(1988) Acta Neurochem. Suppl. 43:94-99; Kanoff, R. B. (1994) J. Am.Osteopath. Assoc. 94:487-493) and osmotic pumps (sold by AlzaCorporation). A particularly preferred mode of administration is via animplantable, externally programmable infusion pump. Suitable infusionpump systems and reservoir systems are also described in U.S. Pat. No.5,368,562 by Blomquist and U.S. Pat. No. 4,731,058 by Doan, developed byPharmacia Deltec Inc.

The method of the invention for altering β-AP aggregation in vivo, andin particular for inhibiting β-AP aggregation, can be usedtherapeutically in diseases associated with abnormal β amyloidaggregation and deposition to thereby slow the rate of β amyloiddeposition and/or lessen the degree of β amyloid deposition, therebyameliorating the course of the disease. In a preferred embodiment, themethod is used to treat Alzheimer's disease (e.g., sporadic or familialAD, including both individuals exhibiting symptoms of AD and individualssusceptible to familial AD). The method can also be usedprophylactically or therapeutically to treat other clinical occurrencesof β amyloid deposition, such as in Down's syndrome individuals and inpatients with hereditary cerebral hemorrhage with amyloidosis-Dutch-type(HCHWA-D). While inhibition of β-AP aggregation is a preferredtherapeutic method, modulators that promote β-AP aggregation may also beuseful therapeutically by allowing for the sequestration of β-AP atsites that do not lead to neurological impairment.

Additionally, abnormal accumulation of β-amyloid precursor protein inmuscle fibers has been implicated in the pathology of sporadic inclusionbody myositis (IBM) (Askana, V. et al. (1996) Proc. Natl. Acad. Sci. USA93:1314-1319; Askanas, V. et al. (1995) Current Opinion in Rheumatology7:486-496). Accordingly, the modulators of the invention can be usedprophylactically or therapeutically in the treatment of disorders inwhich β-AP, or APP, is abnormally deposited at non-neurologicallocations, such as treatment of IBM by delivery of the modulators tomuscle fibers.

VII. Unmodified Aβ Peptides that Inhibit Aggregation of Natural β-AP

In addition to the β-amyloid modulators described hereinbefore in whichan Aβ peptide is coupled to a modifying group, the invention alsoprovides β-amyloid modulators comprised of an unmodified Aβ peptide. Ithas now been discovered that certain portions of natural β-AP can alteraggregation of natural β-APs when contacted with the natural β-APs (seeExample 12). Accordingly, these unmodified Aβ peptides comprise aportion of the natural β-AP sequence (i.e., a portion of βAP₁₋₃₉,βAP₁₋₄₀, βAP₁₋₄₂ and βAP₁₋₄₃). In particular these unmodified Aβpeptides have at least one amino acid deletion compared to βAP₁₋₃₉, theshortest natural β-AP, such that the compound alters aggregation ofnatural β-amyloid peptides when contacted with the natural β-amyloidpeptides. In various embodiments, these unmodified peptide compounds canpromote aggregation of natural β-amyloid peptides, or, more preferably,can inhibit aggregation of natural β-amyloid peptides when contactedwith the natural β-amyloid peptides. Even more preferably, theunmodified peptide compound inhibits aggregation of natural β-amyloidpeptides when contacted with a molar excess amount of natural β-amyloidpeptides (e.g., a 10-fold, 33-fold or 100-fold molar excess amount ofnatural β-AP).

As discussed above, the unmodified peptide compounds of the inventioncomprise an amino acid sequence having at least one amino acid deletioncompared to the amino acid sequence of βAP₁₋₃₉. Alternatively, theunmodified peptide compound can have at least five, ten, fifteen,twenty, twenty-five, thirty or thirty-five amino acids deleted comparedto βAP₁₋₃₉. Still further the unmodified peptide compound can have1-5,1-10, 1-15, 1-20, 1-25, 1-30 or 1-35 amino acids deleted compared toβAP₁₋₃₉. The amino acid deletion(s) may occur at the amino-terminus, thecarboxy-terminus, an internal site, or a combination thereof, of theβ-AP sequence. Accordingly, in one embodiment, an unmodified peptidecompound of the invention comprises an amino acid sequence which has atleast one internal amino acid deleted compared to βAP₁₋₃₉.Alternatively, the unmodified peptide compound can have at least five,ten, fifteen, twenty, twenty-five, thirty or thirty-five internal aminoacids deleted compared to βAP₁₋₃₉. Still further the unmodified peptidecompound can have 1-5,1-10, 1-15, 1-20, 1-25, 1-30 or 1-35 internalamino acids deleted compared to βAP₁₋₃₉. For peptides with internaldeletions, preferably the peptide has an amino terminus corresponding toamino acid residue 1 of natural βAP and a carboxy terminus correspondingto residue 40 of natural βAP and has one or more internal β-AP aminoacid residues deleted (i.e., a non-contiguous Aβ peptide).

In another embodiment, the unmodified peptide compound comprises anamino acid sequence which has at least one N-terminal amino acid deletedcompared to βAP₁₋₃₉ Alternatively, the unmodified peptide compound canhave at least five, ten, fifteen, twenty, twenty-five, thirty orthirty-five N-terminal amino acids deleted compared to βAP₁₋₃₉. Stillfurther the unmodified peptide compound can have 1-5, 1-10, 1-15, 1-20,1-25, 1-30 or 1-35 N-terminal amino acids deleted compared to βAP₁₋₃₉.

In yet another embodiment, the unmodified peptide compound comprises anamino acid sequence which has at least one C-terminal amino acid deletedcompared to βAP₁₋₃₉. Alternatively, the unmodified peptide compound canhave at least five, ten, fifteen, twenty, twenty-five, thirty orthirty-five C-terminal amino acids deleted compared to βAP₁₋₃₉. Stillfurther the unmodified peptide compound can have 1-5,1-10, 1-15, 1-20,1-25, 1-30 or 1-35 C-terminal amino acids deleted compared to βAP₁₋₃₉.

In addition to deletion of amino acids as compared to βAP₁₋₃₉, thepeptide compound can have additional non-β-AP amino acid residues addedto it, for example, at the amino terminus, the carboxy-terminus or at aninternal site. In one embodiment, the peptide compound has at least onenon-β-amyloid peptide-derived amino acid at its N-terminus.Alternatively, the compound can have, for example, 1-3,1-5, 1-7,1-10,1-15 or 1-20 non-β-amyloid peptide-derived amino acid at its N-terminus.In another embodiment, the peptide compound has at least onenon-β-amyloid peptide-derived amino acid at its C-terminus.Alternatively, the compound can have, for example, 1-3,1-5, 1-7,1-10,1-15 or 1-20 non-β-amyloid peptide-derived amino acid at its C-terminus.

In specific preferred embodiments, an unmodified peptide compound of theinvention comprises Aβ₆₋₂₀ (the amino acid sequence of which is shown inSEQ ID NO: 4), Aβ₁₆₋₃₀ (the amino acid sequence of which is shown in SEQID NO: 14), Aβ₁₋₂₀, 26-40 (the amino acid sequence of which is shown inSEQ ID NO: 15) or EEVVHHHHQQ-βAP₁₆₋₄₀ (the amino acid sequence of whichis shown in SEQ ID NO: 16). In the nomenclature used herein,βAP_(1-20, 26-40) represents βAP₁₋₄₀ in which the internal amino acidresidues 21-25 have been deleted.

An unmodified peptide compound of the invention can be chemicallysynthesized using standard techniques such as those described inBodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin(1993) and Grant, G. A (ed.). Synthetic Peptides: A User's Guide, W. H.Freeman and Company, New York (1992). Automated peptide synthesizers arecommercially available (e.g., Advanced ChemTech Model 396;Milligen/Biosearch 9600). Alternatively, unmodified peptide compoundscan be prepared according to standard recombinant DNA techniques using anucleic acid molecule encoding the peptide. A nucleotide sequenceencoding the peptide can be determined using the genetic code and anoligonucleotide molecule having this nucleotide sequence can besynthesized by standard DNA synthesis methods (e.g., using an automatedDNA synthesizer). Alternatively, a DNA molecule encoding an unmodifiedpeptide compound can be derived from the natural β-amyloid precursorprotein gene or cDNA (e.g., using the polymerase chain reaction and/orrestriction enzyme digestion) according to standard molecular biologytechniques.

Accordingly, the invention further provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a β-amyloid peptidecompound, the β-amyloid peptide compound comprising an amino acidsequence having at least one amino acid deletion compared to βAP₁₋₃₉such that the β-amyloid peptide compound alters aggregation of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides.As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules and RNA molecules and may be single-stranded ordouble-stranded, but preferably is double-stranded DNA. The isolatednucleic acid encodes a peptide wherein one or more amino acids aredeleted from the N-terminus, C-terminus and/or an internal site ofβAP₁₋₃₉ as discussed above. In yet other embodiments, the isolatednucleic acid encodes a peptide compound having one or more amino acidsdeleted compared to βAP₁₋₃₉ and further having at least one non-βAPderived amino acid residue added to it, for example, at the aminoterminus, the carboxy-terminus or at an internal site. In specificpreferred embodiments, an isolated nucleic acid molecule of theinvention encodes βAP₆₋₂₀, βAP₁₆₋₃₀, βAP_(1-20, 26-40) orEEVVHHHHQQ-βAP₁₆₋₄₀ (SEQ ID NO: 16).

To facilitate expression of a peptide compound in a host cell bystandard recombinant DNA techniques, the isolated nucleic acid encodingthe peptide is incorporated into a recombinant expression vector.Accordingly, the invention also provides recombinant expression vectorscomprising the nucleic acid molecules of the invention. As used herein,the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” or simply “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors, which serve equivalent functions.

In the recombinant expression vectors of the invention, the nucleotidesequence encoding the peptide compound are operatively linked to one ormore regulatory sequences, selected on the basis of the host cells to beused for expression. The term “operably linked” is intended to mean thatthe sequences encoding the peptide compound are linked to the regulatorysequence(s) in a manner that allows for expression of the peptidecompound. The term “regulatory sequence” is intended to includespromoters, enhancers and other expression control elements (e.g.,polyadenylation signals). Such regulatory sequences are described, forexample, in Goeddel; Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cell, those that direct expression of thenucleotide sequence only in certain host cells (e.g., tissue-specificregulatory sequences) and those that direct expression in a regulatablemanner (e.g., only in the presence of an inducing agent). It will beappreciated by those skilled in the art that the design of theexpression vector may depend on such factors as the choice of the hostcell to be transformed, the level of expression of peptide compounddesired, etc. The expression vectors of the invention can be introducedinto host cells thereby to produce peptide compounds encoded by nucleicacids as described herein.

The recombinant expression vectors of the invention can be designed forexpression of peptide compounds in prokaryotic or eukaryotic cells. Forexample, peptide compounds can be expressed in bacterial cells such asE. coli, insect cells (using baculovirus expression vectors) yeast cellsor mammalian cells. Suitable host cells are discussed further inGoeddel, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Alternatively, the recombinantexpression vector may be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.Examples of vectors for expression in yeast S. cerivisae includepYepSec1 (Baldari et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).Baculovirus vectors available for expression of proteins or peptides incultured insect cells (e.g., Sf 9 cells) include the pAc series (Smithet al., (1983) Mol. Cell. Biol. 3:2156-2165) and the pVL series(Lucklow, V. A., and Summers, M. D., (1989) Virology 170:31-39).Examples of mammalian expression vectors include pCDM8 (Seed, B., (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40.

In addition to the regulatory control sequences discussed above, therecombinant expression vector may contain additional nucleotidesequences. For example, the recombinant expression vector may encode aselectable marker gene to identify host cells that have incorporated thevector. Such selectable marker genes are well known in the art.Moreover, the facilitate secretion of the peptide compound from a hostcell, in particular mammalian host cells, the recombinant expressionvector preferably encodes a signal sequence operatively linked tosequences encoding the amino-terminus of the peptide compound such thatupon expression, the peptide compound is synthesized with the signalsequence fused to its amino terminus. This signal sequence directs thepeptide compound into the secretory pathway of the cell and is thencleaved, allowing for release of the mature peptide compound (i.e., thepeptide compound without the signal sequence) from the host cell. Use ofa signal sequence to facilitate secretion of proteins or peptides frommammalian host cells is well known in the art.

A recombinant expression vector comprising a nucleic acid encoding apeptide compound that alters aggregation of natural β-AP can beintroduced into a host cell to thereby produce the peptide compound inthe host cell. Accordingly, the invention also provides host cellscontaining the recombinant expression vectors of the invention. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein. A host cell may beany prokaryotic or eukaryotic cell. For example, a peptide compound maybe expressed in bacterial cells such as E. coli, insect cells, yeast ormammalian cells. Preferably, the peptide compound is expressed inmammalian cells. In a preferred embodiment, the peptide compound isexpressed in mammalian cells in vivo in a mammalian subject to treatamyloidosis in the subject through gene therapy (discussed furtherbelow). Preferably, the β-amyloid peptide compound encoded by therecombinant expression vector is secreted from the host cell upon beingexpressed in the host cell.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, electroporation, microinjection and viral-mediatedtransfection. Suitable methods for transforming or transfecting hostcells can be found in Sambrook et al. (Molecular Cloning: A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), andother laboratory manuals. Methods for introducing DNA into mammaliancells in vivo are also known in the art and can be used to deliver thevector DNA to a subject for gene therapy purposes (discussed furtherbelow).

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those that confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker may be introduced into a host cell on the samevector as that encoding the peptide compound or may be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A nucleic acid of the invention can be delivered to cells in vivo usingmethods known in the art, such as direct injection of DNA,receptor-mediated DNA uptake or viral-mediated transfection. Directinjection has been used to introduce naked DNA into cells in vivo (seee.g., Acsadi et al. (1991) Nature 332: 815-818; Wolff et al. (1990)Science 247:1465-1468). A delivery apparatus (e.g., a “gene gun”) forinjecting DNA into cells in vivo can be used. Such an apparatus iscommercially available (e.g., from BioRad). Naked DNA can also beintroduced into cells by complexing the DNA to a cation, such aspolylysine, which is coupled to a ligand for a cell-surface receptor(see for example Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621;Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No.5,166,320). Binding of the DNA-ligand complex to the receptorfacilitates uptake of the DNA by receptor-mediated endocytosis.Additionally, a DNA-ligand complex linked to adenovirus capsids whichnaturally disrupt endosomes, thereby releasing material into thecytoplasm can be used to avoid degradation of the complex byintracellular lysosomes (see for example Curiel et al. (1991) Proc.Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl. Acad.Sci. USA 90:2122-2126).

Defective retroviruses are well characterized for use in gene transferfor gene therapy purposes (for a review see Miller, A. D. (1990) Blood76:271). Protocols for producing recombinant retroviruses and forinfecting cells in vitro or in vivo with such viruses can be found inCurrent Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.)Greene Publishing Associates, (1989), Sections 9.10-9.14 and otherstandard laboratory manuals. Examples of suitable retroviruses includepLJ, pZIP, pWE and pEM which are well known to those skilled in the art.Examples of suitable packaging virus lines include ψCrip, ψCre, ψ2 andψAm. Retroviruses have been used to introduce a variety of genes intomany different cell types, including epithelial cells, endothelialcells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitroand/or in vivo (see for example Eglitis, et al. (1985) Science230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

Alternatively, the genome of an adenovirus can be manipulated such thatit encodes and expresses a peptide compound but is inactivated in termsof its ability to replicate in a normal lytic viral life cycle. See forexample Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al.(1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.Suitable adenoviral vectors derived from the adenovirus strain Ad type 5dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are wellknown to those skilled in the art. Recombinant adenoviruses areadvantageous in that they do not require dividing cells to be effectivegene delivery vehicles and can be used to infect a wide variety of celltypes, including airway epithelium (Rosenfeld et al. (1992) citedsupra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad.Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl.Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al. (1992)Proc. Natl. Acad. Sci. USA 89:2581-2584). Additionally, introducedadenoviral DNA (and foreign DNA contained therein) is not integratedinto the genome of a host cell but remains episomal, thereby avoidingpotential problems that can occur as a result of insertional mutagenesisin situations where introduced DNA becomes integrated into the hostgenome (e.g., retroviral DNA).

Adeno-associated virus (AAV) can also be used for delivery of DNA forgene therapy purposes. AAV is a naturally occurring defective virus thatrequires another virus, such as an adenovirus or a herpes virus, as ahelper virus for efficient replication and a productive life cycle. (Fora review see Muzyczka et al. Curr. Topics in Micro. and Immunol. (1992)158:97-129). It is also one of the few viruses that may integrate itsDNA into non-dividing cells, and exhibits a high frequency of stableintegration (see for example Flotte et al. (1992) Am. J. Respir. Cell.Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; andMcLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors containing aslittle as 300 base pairs of AAV can be packaged and can integrate. AnAAV vector such as that described in Tratschin et al (1985) Mol. Cell.Biol. 5:3251-3260 can be used to introduce DNA into cells. A variety ofnucleic acids have been introduced into different cell types using AAVvectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci.USA 81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081;Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al.(1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem.268:3781-3790).

The invention provides a method for treating a subject for a disorderassociated with β-amyloidosis, comprising administering to the subject arecombinant expression vector encoding a β-amyloid peptide compound, thecompound comprising an amino acid sequence having at least one aminoacid deletion compared to βAP₁₋₃₉, such that the β-amyloid peptidecompound is synthesized in the subject and the subject is treated for adisorder associated with β-amyloidosis. Preferably, the disorder isAlzheimer's disease. In one embodiment the recombinant expression vectordirects expression of the peptide compound in neuronal cells. In anotherembodiment, the recombinant expression vector directs expression of thepeptide compound in glial cells. In yet another embodiment, therecombinant expression vector directs expression of the peptide compoundin fibroblast cells.

General methods for gene therapy are known in the art. See for example,U.S. Pat. No. 5,399,346 by Anderson et al. A biocompatible capsule fordelivering genetic material is described in PCT Publication WO 95/05452by Baetge et al. Methods for grafting genetically modified cells totreat central nervous system disorders are described in U.S. Pat. No.5,082,670 and in PCT Publications WO 90/06757 and WO 93/10234, all byGage et al. Isolation and/or genetic modification of multipotent neuralstem cells or neuro-derived fetal cells are described in PCTPublications WO 94/02593 by Anderson et al., WO 94/16718 by Weiss etal., and WO 94/23754 by Major et al. Fibroblasts transduced with geneticmaterial are described in PCT Publication WO 89/02468 by Mulligan et al.Adenovirus vectors for transfering genetic material into cells of thecentral nervous system are described in PCT Publication WO 94/08026 byKahn et al. Herpes simplex virus vectors suitable for treating neuraldisorders are described in PCT Publications WO 94/04695 by Kaplitt andWO 90/09441 by Geller et al. Promoter elements of the glial fibrillaryacidic protein that can confer astrocyte specific expression on a linkedgene or gene fragment, and which thus can be used for expression of Aβpeptides specifically in astrocytes, is described in PCT Publication WO93/07280 by Brenner et al. Furthermore, alternative to expression of anAβ peptide to modulate amyloidosis, an antisense oligonucleotide that iscomplementary to a region of the β-amyloid precursor protein mRNAcorresponding to the peptides described herein can be expressed in asubject to modulate amyloidosis. General methods for expressingantisense oligonucleotides to modulate nervous system disorders aredescribed in PCT Publication WO 95/09236.

Alternative to delivery by gene therapy, a peptide compound of theinvention comprising an amino acid sequence having at least one aminoacid deletion compared to βAP₁₋₃₉ can be delivered to a subject bydirectly administering the peptide compound to the subject as describedfurther herein for the modified peptide compounds of the invention. Thepeptide compound can be formulated into a pharmaceutical compositioncomprising a therapeutically effective amount of the β-amyloid peptidecompound and a pharmaceutically acceptable carrier. The peptide compoundcan be contacted with natural β-amyloid peptides with a β-amyloidpeptide compound such that aggregation of the natural β-amyloid peptidesis inhibited. Moreover, the peptide compound can be administered to thesubject in a therapeutically effective amount such that the subject istreated for a disorder associated with β-amyloidosis, such asAlzheimer's disease.

VIII. Other Embodiments

Although the invention has been illustrated hereinbefore with regard toAβ peptide compounds, the principles described, involving attachment ofa modifying group(s) to a peptide compound, are applicable to anyamyloidogenic protein or peptide as a means to create a modulatorcompound that modulates, and preferably inhibits, amyloid aggregation.Accordingly, the invention provides modulator compounds that can be usedto treat amyloidosis in a variety of forms and clinical settings.

Amyloidosis is a general term used to describe pathological conditionscharacterized by the presence of amyloid. Amyloid is a general termreferring to a group of diverse but specific extracellular proteindeposits which are seen in a number of different diseases. Thoughdiverse in their occurrence, all amyloid deposits have commonmorphologic properties, stain with specific dyes (e.g., Congo red), andhave a characteristic red-green birefringent appearance in polarizedlight after staining. They also share common ultrastructural featuresand common x-ray diffraction and infrared spectra. Amyloidosis can beclassified clinically as primary, secondary, familial and/or isolated.Primary amyloid appears de novo without any preceding disorder.Secondary amyloid is that form which appears as a complication of apreviously existing disorder. Familial amyloid is a geneticallyinherited form found in particular geographic populations. Isolatedforms of amyloid are those that tend to involve a single organ system.

Different amyloids are characterized by the type of protein(s) orpeptide(s) present in the deposit. For example, as describedhereinbefore, amyloid deposits associated with Alzheimer's diseasecomprise the β-amyloid peptide and thus a modulator compound of theinvention for detecting and/or treating Alzheimer's disease is designedbased on modification of the β-amyloid peptide. The identities of theprotein(s) or peptide(s) present in amyloid deposits associated with anumber of other amyloidogenic diseases have been elucidated.Accordingly, modulator compounds for use in the detection and/ortreatment of these other amyloidogenic diseases can be prepared in asimilar fashion to that described herein for β-AP-derived modulators. Invitro assay systems can be established using an amyloidogenic protein orpeptide which forms fibrils in vitro, analogous to the Aβ assaysdescribed herein. Modulators can be identified using such assay systems,based on the ability of the modulator to disrupt the β-sheet structureof the fibrils. Initially, an entire amyloidogenic protein can bemodified or, more preferably, a peptide fragment thereof that is knownto form fibrils in vitro can be modified (e.g., analogous to Aβ₁₋₄₀described herein). Amino acid deletion and substitution analyses canthen be performed on the modified protein or peptide (analogous to thestudies described in the Examples) to delineate an aggregation coredomain that is sufficient, when modified, to disrupt fibril formation.

Non-limiting examples of amyloidogenic proteins or peptides, and theirassociated amyloidogenic disorders, include:

Transthyretin (TTR)—Amyloids containing transthyretin occur in familialamyloid polyneuropathy (Portuguese, Japanese and Swedish types),familial amyloid cardiomyopathy (Danish type), isolated cardiac amyloidand systemic senile amyloidosis. Peptide fragments of transthyretin havebeen shown to form amyloid fibrils in vitro. For example, TTR 10-20 andTTR 105-115 form amyloid-like fibrins in 20-30% acetonitrile/water atroom temperature (Jarvis, J. A., et al. (1994) Int. J. Pept. ProteinRes. 44:388-398). Moreover, familial cardiomyopathy (Danish type) isassociated with mutation of Leu at position 111 to Met, and an analogueof TTR 105-115 in which the wildtype Leu at position 111 has beensubstituted with Met (TTR 105-115Met111) also forms amyloid-like fibrilsin vitro (see e.g., Hermansen, L. F., et al. (1995) Eur. J. Biochem.227:772-779; Jarvis et al. supra). Peptide fragments of TTR that formamyloid fibrils in vitro are also described in Jarvis, J. A., et al.(1993) Biochem. Biophys. Res. Commun. 192:991-998 and Gustavsson, A., etal. (1991) Biochem. Biophys. Res. Commun. 175:1159-1164. A peptidefragment of wildtype or mutated transthyretin that forms amyloid fibrilscan be modified as described herein to create a modulator of amyloidosisthat can be used in the detection or treatment of familial amyloidpolyneuropathy (Portuguese, Japanese and Swedish types), familialamyloid cardiomyopathy (Danish type), isolated cardiac amyloid orsystemic senile amyloidosis.

Prion Protein (PrP)—Amyloids in a number of spongiform encephalopathies,including scrapie in sheep, bovine spongiform encephalopathy in cows andCreutzfeldt-Jakob disease (CJ) and Gerstmann-Straussler-Scheinkersyndrome (GSS) in humans, contain PrP. Limited proteolysis of PrPSc (theprion protein associated with scrapie) leads to a 27-30 kDa fragment(PrP27-30) that polymerizes into rod-shaped amyloids (see e.g., Pan, K.M., et al. (1993) Proc. Natl. Acad. Sci. USA 90:10962-10966; Gasset, M.,et al. (1993) Proc. Natl. Acad. Sci. USA 90:1-5). Peptide fragments ofPrP from humans and other mammals have been shown to form amyloidfibrils in vitro. For example, polypeptides corresponding to sequencesencoded by normal and mutant alleles of the PRNP gene (encoding theprecursor of the prion protein involved in CJ), in the regions of codon178 and codon 200, spontaneously form amyloid fibrils in vitro (seee.g., Goldfarb, L. G., et al. (1993) Proc. Natl. Acad. Sci. USA90:4451-4454). A peptide encompassing residues 106-126 of human PrP hasbeen reported to form straight fibrils similar to those extracted fromGSS brains, whereas a peptide encompassing residues 127-147 of human PrPhas been reported to form twisted fibrils resembling scrapie-associatedfibrils (Tagliavini, F., et al. (1993) Proc. Natl. Acad. Sci. USA90:9678-9682). Peptides of Syrian hamster PrP-encompassing residues109-122, 113-127, 113-120, 178-191 or 202-218 have been reported to formamyloid fibrils, with the most amyloidogenic peptide beingAla-Gly-Ala-Ala-Ala-Ala-Gly-Ala (SEQ ID NO: 17), which corresponds toresidues 113-120 of Syrian hamster PrP but which is also conserved inPrP from other species (Gasset, M., et al. (1992) Proc. Natl. Acad. Sci.USA 89:10940-10944). A peptide fragment of PrP that forms amyloidfibrils can be modified as described herein to create a modulator ofamyloidosis that can be used in the detection or treatment of scrapie,bovine spongiform encephalopathy, Creutzfeldt-Jakob disease orGerstmann-Straussler-Scheinker syndrome.

Islet Amyloid Polypeptide (IAPP, also known as amylin)—Amyloidscontaining IAPP occur in adult onset diabetes and insulinoma. IAPP is a37 amino acid polypeptide formed from an 89 amino acid precursor protein(see e.g., Betsholtz, C., et al. (1989) Exp. Cell. Res. 183:484-493;Westermark, P., et al. (1987) Proc. Natl. Acad. Sci. USA 84:3881-3885).A peptide corresponding to IAPP residues 20-29 has been reported to formamyloid-like fibrils in vitro, with residues 25-29, having the sequenceAla-Ile-Leu-Ser-Ser (SEQ ID NO: 18), being strongly amyloidogenic(Westermark, P., et al. (1990) Proc. Natl. Acad. Sci. USA 87:5036-5040;Glenner, G. G., et al. (1988) Biochem. Biophys. Res. Commun.155:608-614). A peptide fragment of IAPP that forms amyloid fibrils canbe modified as described herein to create a modulator of amyloidosisthat can be used in the detection or treatment of adult onset diabetesor insulinoma.

Atrial Natriuretic Factor (ANF)—Amyloids containing ANF are associatedwith isolated atrial amyloid (see e.g., Johansson, B., et al. (1987)Biochem. Biophys. Res. Commun. 148:1087-1092). ANF corresponds to aminoacid residues 99-126 (proANF99-126) of the ANF prohormone (proANPI-126)(Pucci, A., et al. (1991) J. Pathol. 165:235-241). ANF, or a fragmentthereof, that forms amyloid fibrils can be modified as described hereinto create a modulator of amyloidosis that can be used in the detectionor treatment of isolated atrial amyloid.

Kappa or Lambda Light Chain—Amyloids containing kappa or lambda lightchains are associated idiopathic (primary) amyloidosis, myeloma ormacroglobulinemia-associated amyloidosis, and primary localizedcutaneous nodular amyloidosis associated with Sjogren's syndrome. Thestructure of amyloidogenic kappa and lambda light chains, includingamino acid sequence analysis, has been characterized (see e.g., Buxbaum,J. N., et al. (1990) Ann. Intern. Med. 112:455-464; Schormann, N., etal. (1995) Proc. Natl. Acad. Sci. USA 92:9490-9494; Hurle, M. R., et al.(1994) Proc. Natl. Acad. Sci. USA 91:5446-5450; Liepnieks, J. J., et al.(1990) Mol. Immunol. 27:481-485; Gertz, M. A., et al. (1985) Scand. J.Immunol. 22:245-250; Inazumi, T., et al. (1994) Dermatology189:125-128). Kappa or lambda light chains, or a peptide fragmentthereof that forms amyloid fibrils, can be modified as described hereinto create a modulator of amyloidosis that can be used in the detectionor treatment of idiopathic (primary) amyloidosis, myeloma ormacroglobulinemia-associated amyloidosis or primary localized cutaneousnodular amyloidosis associated with Sjogren's syndrome.

Amyloid A—Amyloids containing the amyloid A protein (AA protein),derived from serum amyloid A, are associated with reactive (secondary)amyloidosis (see e.g., Liepnieks, J. J., et al. (1995) Biochim. Biophys.Acta 1270:81-86), familial Mediterranean Fever and familial amyloidnephropathy with urticaria and deafness (Muckle-Wells syndrome) (seee.g., Linke, R. P., et al. (1983) Lab. Invest. 48:698-704). Recombinanthuman serum amyloid A forms amyloid-like fibrils in vitro (Yamada, T.,et al. (1994) Biochim. Biophys. Acta 1226:323-329) and circulardichroism studies revealed a predominant β sheet/turn structure(McCubbin, W. D., et al. (1988) Biochem J. 256:775-783). Serum amyloidA, amyloid A protein or a fragment thereof that forms amyloid fibrilscan be modified as described herein to create a modulator of amyloidosisthat can be used in the detection or treatment of reactive (secondary)amyloidosis, familial Mediterranean Fever and familial amyloidnephropathy with urticaria and deafness (Muckle-Wells syndrome).

Cystatin C—Amyloids containing a variant of cystatin C are associatedwith hereditary cerebral hemorrhage with amyloidosis of Icelandic type.The disease is associated with a leucine to glycine mutation at position68 and cystatin C containing this mutation aggregates in vitro(Abrahamson, M. and Grubb, A. (1994) Proc. Natl. Acad. Sci. USA91:1416-1420). Cystatin C or a peptide fragment thereof that formsamyloid fibrils can be modified as described herein to create amodulator of amyloidosis that can be used in the detection or treatmentof hereditary cerebral hemorrhage with amyloidosis of Icelandic type.

β2 microglobulin—Amyloids containing β2 microglobulin (β2M) are a majorcomplication of long term hemodialysis (see e.g., Stein, G., et al.(1994) Nephrol. Dial. Transplant. 9:48-50; Floege, J., et al. (1992)Kidney Int. Suppl. 38:S78-S85; Maury, C. P. (1990) Rheumatol. Int.10:1-8). The native β2M protein has been shown to form amyloid fibrilsin vitro (Connors, L. H., et al. (1985) Biochem. Biophys. Res. Commun.131:1063-1068; Ono, K., et al. (1994) Nephron 66:404-407). β2M, or apeptide fragment thereof that forms amyloid fibrils, can be modified asdescribed herein to create a modulator of amyloidosis that can be usedin the detection or treatment of amyloidosis associated with long termhemodialysis.

Apolipoprotein A-I (ApoA-I)—Amyloids containing variant forms of ApoA-Ihave been found in hereditary non-neuropathic systemic amyloidosis(familial amyloid polyneuropathy III). For example, N-terminal fragments(residues 1-86, 1-92 and 1-93) of an ApoA-I variant having a Trp to Argmutation at position 50 have been detected in amyloids (Booth, D. R., etal. (1995) QJM 88:695-702). In another family, a leucine to argininemutation at position 60 was found (Soutar, A. K., et al. (1992) Proc.Natl. Acad. Sci. USA 89:7389-7393). ApoA-I or a peptide fragment thereofthat forms amyloid fibrils can be modified as described herein to createa modulator of amyloidosis that can be used in the detection ortreatment of hereditary non-neuropathic systemic amyloidosis.

Gelsolin—Amyloids containing variants of gelsolin are associated withfamilial amyloidosis of Finnish type. Synthetic gelsolin peptides thathave sequence homology to wildtype or mutant gelsolins and that formamyloid fibrils in vitro are reported in Maury, C. P. et al. (1994) Lab.Invest. 70:558-564. A nine residue segment surrounding residue 187(which is mutated in familial gelsolin amyloidosis) was defined as anamyloidogenic region (Maury, et al., supra; see also Maury, C. P., etal. (1992) Biochem. Biophys. Res. Commun. 183:227-231; Maury, C. P.(1991) J. Clin. Invest. 87:1195-1199). Gelsolin or a peptide fragmentthereof that forms amyloid fibrils can be modified as described hereinto create a modulator of amyloidosis that can be used in the detectionor treatment of familial amyloidosis of Finnish type.

Procalcitonin or calcitonin—Amyloids containing procalcitonin,calcitonin or calcitonin-like immunoreactivity have been detected inamyloid fibrils associated with medullary carcinoma of the thyroid (seee.g., Butler, M. and Khan, S. (1986) Arch. Pathol. Lab. Med.110:647-649; Sletten, K., et al. (1976) J. Exp. Med. 143:993-998).Calcitonin has been shown to form a nonbranching fibrillar structure invitro (Kedar, I., et al. (1976) Isr. J. Med. Sci. 12:1137-1140).Procalcitonin, calcitonin or a fragment thereof that forms amyloidfibrils can be modified as described herein to create a modulator ofamyloidosis that can be used in the detection or treatment ofamyloidosis associated with medullary carcinoma of the thyroid.

Fibrinogen—Amyloids containing a variant form of fibrinogen alpha-chainhave been found in hereditary renal amyloidosis. An arginine to leucinemutation at position 554 has been reported in amyloid fibril proteinisolated from postmortem kidney of an affected individual (Benson, M.D., et al. (1993) Nature Genetics 3:252-255). Fibrinogen alpha-chain ora peptide fragment thereof that forms amyloid fibrils can be modified asdescribed herein to create a modulator of amyloidosis that can be usedin the detection or treatment of fibrinogen-associated hereditary renalamyloidosis.

Lysozyme—Amyloids containing a variant form of lysozyme have been foundin hereditary systemic amyloidosis. In one family the disease wasassociated with a threonine to isoleucine mutation at position 56,whereas in another family the disease was associated with a histidine toaspartic acid mutation at position 67 (Pepys, M. B., et al. (1993)Nature 362:553-557). Lysozyme or a peptide fragment thereof that formsamyloid fibrils can be modified as described herein to create amodulator of amyloidosis that can be used in the detection or treatmentof lysozyme-associated hereditary systemic amyloidosis.

This invention is further illustrated by the following examples whichshould not be construed as limiting. A modulator's ability to alter theaggregation of β-amyloid peptide in the assays described below arepredictive of the modulator's ability to perform the same function invivo. The contents of all references, patents and published patentapplications cited throughout this application are hereby incorporatedby reference.

Example 1 Construction of β-Amyloid Modulators

A β-amyloid modulator composed of an amino-terminally biotinylatedβ-amyloid peptide of the amino acid sequence:

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV

(positions 1 to 40 of SEQ ID NO: 1) was prepared by solid-phase peptidesynthesis using an N^(α)-9-fluorenylmethyloxycarbonyl (FMOC)-basedprotection strategy as follows. Starting with 2.5 mmoles ofFMOC-Val-Wang resin, sequential additions of each amino acid wereperformed using a four-fold excess of protected amino acids,1-hydroxybenzotriazole (HOBt) and diisopropyl carbodiimide (DIC).Recouplings were performed when necessary as determined by ninhydrintesting of the resin after coupling. Each synthesis cycle was minimallydescribed by a three minute deprotection (25%piperidine/N-methyl-pyrrolidone (NMP)), a 15 minute deprotection, fiveone minute NMP washes, a 60 minute coupling cycle, five NMP washes and aninhydrin test. To a 700 mg portion of the fully assembledpeptide-resin, biotin (obtained commercially from Molecular Probes,Inc.) was substituted for an FMOC-amino acid was coupled by the aboveprotocol. The peptide was removed from the resin by treatment withtrifluoroacetic acid (TFA) (82.5%), water (5%), thioanisole (5%), phenol(5%), ethanedithiol (2.5%) for two hours followed by precipitation ofthe peptide in cold ether. The solid was pelleted by centrifugation(2400 rpm×10 min.), and the ether decanted. It was resuspended in ether,pelleted and decanted a second time. The solid was dissolved in 10%acetic acid and lyophilized to dryness to yield 230 mg of crudebiotinylated peptide. 60 mg of the solid was dissolved in 25%acetonitrile (ACN)/0.1% TFA and applied to a C18 reversed phase highperformance liquid chromatography (HPLC) column. Biotinyl βAP₁₋₄₀ waseluted using a linear gradient of 30-45% acetonitrile/0.1% TFA over 40minutes. One primary fraction (4 mg) and several side fractions wereisolated. The main fraction yielded a mass spectrum of 4556(matrix-assisted laser desorption ionization—time of flight) whichmatches the theoretical (4555) for this peptide.

A β-amyloid modulator composed of an amino-terminally biotinylatedβ-amyloid peptide of the amino acid sequence:

DAEFRHDSGYEVHHQ

(positions 1 to 15 of SEQ ID NO: 1) was prepared on an Advanced ChemTechModel 396 multiple peptide synthesizer using an automated protocolestablished by the manufacturer for 0.025 mmole scale synthesis. Doublecouplings were performed on all cycles using2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU)/N,N-diisopropylethylamine (DIEA)/HOBt/FMOC-AA in four-fold excessfor 30 minutes followed by DIC/HOBt/FMOC-AA in four-fold excess for 45minutes. The peptide was deprotected and removed from the resin bytreatment with TFA/water (95%/5%) for three hours and precipitated withether as described above. The pellet was resuspended in 10% acetic acidand lyophilized. The material was purified by a preparative HPLC using15%-40% acetonitrile over 80 minutes on a Vydac C18 column (21×250 mm).The main isolate eluted as a single symmetrical peak when analyzed byanalytical HPLC and yielded the expected molecular weight when analyzedby electrospray mass spectrometry. Result=2052.6 (2052 theoretical).

β-amyloid modulator compounds comprising other regions of the β-AP aminoacid sequence (e.g., an Aβ aggregation core domain) were similarlyprepared using the synthesis methods described above. Moreover,modulators comprising other amyloidogenic peptides can be similarlyprepared.

Example 2 Inhibition of β-Amyloid Aggregation by Modulators

The ability of β-amyloid modulators to inhibit the aggregation ofnatural β-AP when combined with the natural β-AP was examined in aseries of aggregation assays. Natural β-AP(β-AP₁₋₄₀) was obtainedcommercially from Bachem (Torrance, Calif.). Amino-terminallybiotinylated β-AP modulators were prepared as described in Example 1.

A. Optical Density Assay

In one assay, β-AP aggregation was measured by determining the increasein turbidity of a solution of natural β-AP over time in the absence orpresence of various concentrations of the modulator. Turbidity of thesolution was quantitated by determining the optical density at 400 nm(A_(400 nm)) of the solution over time.

The aggregation of natural β-AP in the absence of modulator wasdetermined as follows. β-AP₁₋₄₀ was dissolved in hexafluoro isopropanol(HFIP; Aldrich Chemical Co., Inc.) at 2 mg/ml. Aliquots of the HFIPsolution (87 μl) were transferred to individual 10 mm×75 mm test tubes.A stream of argon gas was passed through each tube to evaporate theHFIP. To the resulting thin film of peptide, dimethylsulfoxide (DMSO;Aldrich Chemical Co., Inc.) (25 μl) was added to dissolve the peptide. A2 mm×7 mm TEFLON™-coated magnetic stir bar was added to each tube.Buffer (475 μL of 100 mM NaCl, 10 mM sodium phosphate, pH 7.4) was addedto the DMSO solution with stirring. The resulting mixture was stirredcontinuously and the optical density was monitored at 400 nm to observethe formation of insoluble peptide aggregates.

Alternatively, β-AP₁₋₄₀ was dissolved in DMSO as described above at 1.6mM (6.9 mg/ml) and aliquots (25 μl) were added to stirred buffer (475μl), followed by monitoring of absorbance at 400 nm.

For inhibition studies in which a β-amyloid modulator was dissolved insolution together with the natural β-AP, the modulators were dissolvedin DMSO either with or without prior dissolution in HFIP. Thesecompounds were then added to buffer with stirring, followed by additionof β-AP₁₋₄₀ in DMSO. Alternatively, HFIP solutions of modulators werecombined with β-AP₁₋₄₀ in HFIP followed by evaporation and redissolutionof the mixture in DMSO. Buffer was then added to the DMSO solution toinitiate the assay. The amino-terminally biotinylated β-amyloid peptidemodulators N-biotinyl-βAP₁₋₄₀, and N-biotinyl-βAP₁₋₁₅ were tested atconcentrations of 1% and 5% in the natural β-AP₁₋₄₀ solution.

A representative example of the results is shown graphically in FIG. 1,which depicts the inhibition of aggregation of natural β-AP₁₋₄₀ byN-biotinyl-βAP₁₋₄₀. In the absence of the modulator, the optical densityof the natural β-AP solution showed a characteristic sigmoidal curve,with a lag time prior to aggregation (approximately 3 hours in FIG. 1)in which the A_(400 nm) was low, followed by rapid increase in theA_(400 nm), which quickly reached a plateau level, representingaggregation of the natural β amyloid peptides. In contrast, in thepresence of as little as 1% of the N-biotinyl-βAP₁₄₀ modulator,aggregation of the natural β amyloid peptides was markedly inhibited,indicated by an increase in the lag time, a decrease in the slope ofaggregation and a decrease in the plateau level reached for theturbidity of the solution (see FIG. 1). N-biotinyl-βAP₁₋₄₀ at aconcentration of 5% similarly inhibited aggregation of the natural βamyloid peptide. Furthermore, similar results were observed whenN-biotinyl-βAP₁₋₁₅ was used as the modulator. These results demonstratethat an N-terminally biotinylated β-AP modulator can effectively inhibitthe aggregation of natural β amyloid peptides, even when the natural βamyloid peptides are present at as much as a 100-fold molar excessconcentration.

B. Fluorescence Assay

In a second assay, β-AP aggregation was measured using a fluorometricassay essentially as described in Levine, H. (1993) Protein Science2:404-410. In this assay, the dye thioflavine T (ThT) is contacted withthe β-AP solution. Association of ThT with aggregated β-AP, but notmonomeric or loosely associated β-AP, gives rise to a new excitation(ex) maximum at 450 nm and an enhanced emission (em) at 482 nm, comparedto the 385 nm (ex) and 445 nm (em) for the free dye. β-AP aggregationwas assayed by this method as follows. Aliquots (2.9 μl) of thesolutions used in the aggregation assays as described above in section Awere removed from the samples and diluted in 200 μl of potassiumphosphate buffer (50 mM, pH 7.0) containing thioflavin T (10 μM;obtained commercially from Aldrich Chemical Co., Inc.). Excitation wasset at 450 nm and emission was measured at 482 mm. Similar to theresults observed with the optical density assay described above insection A, as little as 1% of the N-biotinylated β-AP modulators waseffective at inhibiting the aggregation of natural β amyloid peptidesusing this fluorometric assay.

C. Static Aggregation Assay

In a third assay, β-AP aggregation was measured by visualization of thepeptide aggregates using SDS-polyacrylamide gel electrophoresis(SDS-PAGE). In this assay, β-AP solutions were allowed to aggregate overa period of time and then aliquots of the reaction were run on astandard SDS-PAGE gel. Typical solution conditions were 200 μM ofβ-AP₁₋₄₀ in PBS at 37° C. for 8 days or 200 μM β-AP₁₋₄₀ in 0.1 M sodiumacetate at 37° C. for 3 days. The peptide aggregates were visualized byCoomassie blue staining of the gel or, for β-AP solutions that includeda biotinylated S-AP modulator, by western blotting of a filter preparedfrom the gel with a streptavidin-peroxidase probe, followed by astandard peroxidase assay. The β-AP aggregates are identifiable as highmolecular weight, low mobility bands on the gel, which are readilydistinguishable from the low molecular weight, high mobility β-APmonomer or dimer bands.

When natural β-AP₁₋₄₀ aggregation was assayed by this method in theabsence of any β amyloid modulators, high molecular weight aggregateswere readily detectable on the gel. In contrast, whenN-biotinyl-β-AP₁₋₄₀ modulator self-aggregation was assayed (i.e.,aggregation of the N-biotinyl peptide alone, in the absence of anynatural β-AP), few if any high molecular weight aggregates wereobserved, indicating that the ability of the modulator to self-aggregateis significantly reduced compared to natural β-AP. Finally, whenaggregation of a mixture of natural β-AP₁₋₄₀ and N-biotinylated β-AP₁₋₄₀was assayed by this method, reduced amounts of the peptide mixtureassociated into high molecular weight aggregates, thus demonstratingthat the β amyloid modulator is effective at inhibiting the aggregationof the natural β amyloid peptides.

Example 3 Neurotoxicity Analysis of β-Amyloid Modulators

The neurotoxicity of the β-amyloid modulators is tested in a cell-basedassay using the neuronal precursor cell line PC-12, or primary neuronalcells, and the viability indicator3,(4,4-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT). (SeeShearman, M. S. et al. (1994) Proc. Natl. Acad. Sci. USA 91:1470-1474;Hansen, M. B. et al. (1989) J. Immun. Methods 119:203-210). PC-12 is arat adrenal pheochromocytoma cell line and is available from theAmerican Type Culture Collection, Rockville, Md. (ATCC CRL 1721). MTT(commercially available from Sigma Chemical Co.) is a chromogenicsubstrate that is converted from yellow to blue in viable cells, whichcan be detected spectrophotometrically.

To test the neurotoxicity of a β-amyloid modulator (either alone orcombined with natural β-AP), cells first are plated in 96-well plates at7,000-10,000 cells/well and allowed to adhere by overnight culture at37° C. Serial dilutions of freshly dissolved or “aged” modulators(either alone or combined with natural N-AP) in phosphate bufferedsaline (PBS) are added to the wells in triplicate and incubation iscontinued for two or more days. Aged modulators are prepared byincubating an aqueous solution of the modulator at 37° C. undisturbedfor a prolonged period (e.g., five days or more). For the final twohours of exposure of the cells to the modulator preparation, MTT isadded to the media to a final concentration of 1 mg/ml and incubation iscontinued at 37° C. Following the two hour incubation with MTT, themedia is removed and the cells are lysed in isopropanol/0.4N HCl withagitation. An equal volume of PBS is added to each well and theabsorbance of each well at 570 nm is measured to quantitate viablecells. Alternatively, MTT is solubilized by addition of 50% N,N-dimethylformamide/20% sodium dodecyl sulfate added directly to the media in thewells and viable cells are likewise quantitated by measuring absorbanceat 570 nm. The relative neurotoxicity of a β-amyloid modulator (eitheralone or in combination with natural β-AP) is determined by comparisonto natural β-AP alone (e.g., β1-40, β1-42), which exhibits neurotoxicityin this assay and thus can serve as a positive control.

Example 4 Syntheses of Additional Modified β-Amyloid Peptide Compounds

In this example, a series of modified β-APs, having a variety ofN-terminal or random side chain modifications were synthesized.

A series of N-terminally modified p-amyloid peptides was synthesizedusing standard methods. Fully-protected resin-bound peptidescorresponding to Aβ(1-15) and Aβ(1-40) were prepared as described inExample 1 on Wang resin to eventually afford carboxyl terminal peptideacids. Small portions of each peptide resin (13 and 20 μmoles,respectively) were aliquoted into the wells of the reaction block of anAdvanced ChemTech Model 396 Multiple Peptide Synthesizer. The N-terminalFMOC protecting group of each sample was removed in the standard mannerwith 25% piperidine in NMP followed by extensive washing with NMP. Theunprotected N-terminal a-amino group of each peptide-resin sample wasmodified using one of the following methods:

Method A, coupling of modifying reagents containing free carboxylic acidgroups: The modifying reagent (five equivalents) was predissolved inNMP, DMSO or a mixture of these two solvents. HOBT and DIC (fiveequivalents of each reagent) were added to the dissolved modifier andthe resulting solution was added to one equivalent of free-aminopeptide-resin. Coupling was allowed to proceed overnight, followed bywashing. If a ninhydrin test on a small sample of peptide-resin showedthat coupling was not complete, the coupling was repeated using1-hydroxy-7-azabenzotriazole (HOAt) in place of HOBt.

Method B, coupling of modifying reagents obtained in preactivated forms:The modifying reagent (five equivalents) was predissolved in NMP, DMSOor a mixture of these two solvents and added to one equivalent ofpeptide-resin. Diisopropylethylamine (DIEA; six equivalents) was addedto the suspension of activated modifier and peptide-resin. Coupling wasallowed to proceed overnight, followed by washing. If a ninhydrin teston a small sample of peptide-resin showed that coupling was notcomplete, the coupling was repeated.

After the second coupling (if required) the N-terminally modifiedpeptide-resins were dried at reduced pressure and cleaved from the resinwith removal of side-chain protecting groups as described in Example 1.Analytical reversed-phase HPLC was used to confirm that a major productwas present in the resulting crude peptides which were purified usingMillipore Sep-Pak cartridges or preparative reverse-phase HPLC. Massspectrometry was used to confirm the presence of the desired compound inthe product.

Method A was used to couple N-acetylneuraminic acid, cholic acid,trans-4-cotininecarboxylic acid, 2-imino-1-imidazolidineacetic acid,(S)-(−)-indoline-2-carboxylic acid, (−)-menthoxyacetic acid,2-norbornaneacetic acid, γ-oxo-5-acenaphthenebutyric acid,(−)-2-oxo-4-thiazolidinecarboxylic acid, and tetrahydro-3-furoic acid.Method B was used to couple 2-iminobiotin-N-hydroxysuccinimide ester,diethylenetriaminepentaacetic dianhydride, 4-morpholinecarbonylchloride, 2-thiopheneacetyl chloride, and 2-thiophenesulfonyl chloride.

In a manner similar to the construction of N-terminally modifiedAβ(1-15) and Aβ(1-40) peptides described above, N-fluoresceinyl Aβ(1-15)and Aβ(1-40) were prepared in two alternative manners using thepreactivated reagents 5-(and 6)-carboxyfluorescein succinimidyl esterand fluorescein-5-isothiocyanate (FITC Isomer I). Both reagents wereobtained from Molecular Probes Inc. Couplings were performed using fourequivalents of reagent per equivalent of peptide-resin with DIEA addedto make the reaction solution basic to wet pH paper. Couplings of eachreagent to Aβ(1-15)-resin appeared to be complete after a singleovernight coupling. Coupling to Aβ(1-40)-resin was slower as indicatedby a positive ninhydrin test and both reagents were recoupled to thispeptide-resin overnight in tetrahydrofuran-NMP (1:2 v/v). The resultingN-terminally modified peptide-resins were cleaved, deprotected andpurified as described in Example A.

In addition to the N-fluoresceinyl Aβ peptides described above, aβ-amyloid modulator comprised of random modification of Aβ(1-40) withfluorescein was prepared. Aβ(1-40) purchased from Bachem was dissolvedin DMSO at approximately 2 mg/mL. 5-(and-6)-Carboxyfluorescein purchasedfrom Molecular Probes was added in a 1.5 molar excess and DIEA was addedto make the solution basic to wet pH paper. The reaction was allowed toproceed for 1 hour at room temperature and was then quenched withtriethanolamine. The product was added to assays as this crude mixture.

β-amyloid modulator compounds comprising other regions of the β-AP aminoacid sequence (e.g., an Aβ aggregation core domain) were similarlyprepared using the synthesis methods described above. Moreover,modulators comprising other amyloidogenic peptides can be similarlyprepared.

Example 5 Identification of Additional β-Amyloid Modulators

In this Example, two assays of Aβ aggregation were used to identifyβ-amyloid modulators which can inhibit this process.

The first assay is referred to as a seeded static assay (SSA) and wasperformed as follows:

To prepare a solution of Aβ monomer, the appropriate quantity ofAβ(1-40) peptide (Bachem) was weighed out on a micro-balance (the amountwas corrected for the amount of water in the preparation, which,depending on lot number, was 20-30% w/w). The peptide was dissolved in1/25 volume of dimethylsulfoxide (DMSO), followed by water to ½ volumeand ½ volume 2× PBS (10× PBS: NaCl 137 mM, KCl 2.7 mM Na₂HPO₄.7H₂O 4.3mM, KH₂PO₄ 1.4 mM pH 7.2) to a final concentration of 200 μM.

To prepare a stock seed, 1 ml of the above Aβ monomer preparation, wasincubated for 8 days at 37° C. and sheared sequentially through an 18,23, 26 and 30 gauge needle 25, 25, 50, and 100 times respectively. 2 μlsamples of the sheared material was taken for fluorescence measurementsafter every 50 passes through the 30 gauge needle until the fluorescenceunits (FU) had plateaued (approx. 100-150×).

To prepare a candidate inhibitor, the required amount of candidateinhibitor was weighed out and the stock dissolved in 1×PBS to a finalconcentration of 1 mM (10× stock). If insoluble, it was dissolved in1/10 volume of DMSO and diluted in 1×PBS to 1 mM. A further 1/10dilution was also prepared to test each candidate at both 100 μM and 10μM.

For the aggregation assay, each sample was set up in triplicate [50 μlof 200 μM monomer, 125 FU sheared seed (variable quantity dependent onthe batch of seed, routinely 3-6 μl), 10 μl of 10× inhibitor solution,final volume made up to 100 μl with 1×PBS]. Two concentrations of eachinhibitor were tested 100 μM and 10 μM, equivalent to a 1:1 and a 1:10molar ratio of monomer to inhibitor. The controls included an unseededreaction to confirm that the fresh monomer contained no seed, and aseeded reaction in the absence of inhibitor, as a reference to compareagainst putative inhibitors. The assay was incubated at 37° C. for 6 h,taking 2 μl samples hourly for fluorescence measurements. To measurefluorescence, a 2 μl sample of Aβ was added to 400 μl of Thioflavin-Tsolution (50 mM Potassium Phosphate 10 mM Thioflavin-T pH 7.5). Thesamples were vortexed and the fluorescence was read in a 0.5 ml microquartz cuvette at EX 450 nm and EM 482 nm (Hitachi 4500 Fluorimeter).β-aggregation results in enhanced emission of Thioflavin-T. Accordingly,samples including an effective inhibitor compound exhibit reducedemission as compared to control samples without the inhibitor compound.

The second assay is referred to as a shaken plate aggregation assay andwas performed as follows:

Aβ(1-40) peptide from Bachem (Torrance, Calif.) was dissolved in HFIP(1,1,1,3,3,3-Hexafluoro-2-propanol; Aldrich 10,522-8) at a concentrationof 2 mg peptide/ml and incubated at room temperature for 30 min. HFIPsolubilized peptide was sonicated in a waterbath sonicator for 5 min athighest setting, then evaporated to dryness under a stream of argon. Thepeptide film was resuspended in anhydrous dimethylsulfoxide (DMSO) at aconcentration of 6.9 mg/ml, sonicated for 5 min as before, then filteredthrough a 0.2 micron nylon syringe filter (VWR cat. No. 28196-050).Candidate inhibitors were dissolved directly in DMSO, generally at amolar concentration 4 times that of the Aβ(1-40) peptide.

Candidates were assayed in triplicate. For each candidate to be tested,4 parts Aβ(1-40) peptide in DMSO were combined with 1 part candidateinhibitor in DMSO in a glass vial, and mixed to produce a 1:1 molarratio of Aβ peptide to candidate. For different molar ratios, candidateswere diluted with DMSO prior to addition to Aβ(1-40), in order to keepthe final DMSO and Aβ(1-40) concentrations constant. Into an ultra lowbinding 96 well plate (Corning Costar cat. No. 2500, Cambridge Mass.)100 μl PTL buffer (150 mM NaCl, 10 mM NaH₂PO₄; pH 7.4) was aliquottedper well. For each candidate, 10 μl of peptide mixture in DMSO wasaliquotted into each of three wells containing buffer. The covered platewas vigorously vortexed on a plate shaker at high speed for 30 seconds.An additional 100 μl of PTL buffer was added to each well and again theplate was vortexed vigorously for 30 sec. Absorbance at 405 nm wasimmediately read in a plate reader for a baseline reading. The plate wasreturned to the plate shaker and vortexed at moderate speed for 5 hoursat room temperature, with absorbance readings taken at 15-20 minintervals. Increased absorbance indicated aggregation. Accordingly,effective inhibitor compounds cause a decrease in absorbance in the testsample as compared to a control sample without the inhibitor compound.

Representative results of the static seeded assay and shaken plate assaywith preferred β-amyloid modulators are shown below in Table I.

TABLE I Effect Effect in in Seeded Candidate Aβ Amino Modifying shakenplate Static Inhibitor Acids Reagent assay Assay* 174 Aβ1-15 Cholic acidComplete ++ inhibition at 100% conc 176 Aβ1-15 Diethylene- Decreased ++triamine penta Plateau acetic acid 180 Aβ1-15 (−)-Menthoxy None ++acetic acid 190 Aβ1-15 Fluorescein Decreased ++ carboxylic acid Plateau(FICO) 220 Aβ16-40 NH₂- Complete ++ mutant EVHHHHQQK- inhibition at[Aβ(16-40)]- 100%, COOH increased (SEQ ID NO: 16) lag at 10% 224 Aβ1-40F₁₉F₂₀−>T₁₉T₂₀ Increased lag ++ mutant 233 A6β-20 Acetic acidaccelerated ++ aggregation at 10% conc *++ = A strong inhibitor ofaggregation. The rate of aggregation in the presence of the inhibitorwas decreased compared to the control by at least 30-50%

These results indicate that β-APs modified by a wide variety ofN-terminal modifying groups are effective at modulating β-amyloidaggregation.

Example 6 Additional β-Amyloid Aggregation Assays

Most preferably, the ability of β-amyloid modulator compounds tomodulate (e.g., inhibit or promote) the aggregation of natural β-AP whencombined with the natural β-AP is examined in one or both of theaggregation assays described below. Natural β-AP (β-AP₁₋₄₀) for use inthe aggregation assays is commercially available from Bachem (Torrance,Calif.).

A. Nucleation Assay

The nucleation assay is employed to determine the ability of testcompounds to alter (e.g. inhibit) the early events in formation of β-APfibers from monomeric β-AP. Characteristic of a nucleated polymerizationmechanism, a lag time is observed prior to nucleation, after which thepeptide rapidly forms fibers as reflected in a linear rise in turbidity.The time delay before polymerization of β-AP monomer can be quantifiedas well as the extent of formation of insoluble fiber by lightscattering (turbidity). Polymerization reaches equilibrium when themaximum turbidity reaches a plateau. The turbidity of a solution ofnatural β-AP in the absence or presence of various concentrations of aβ-amyloid modulator compound is determined by measuring the apparentabsorbance of the solution at 405 nm (A_(405 nm)) over time. Thethreshold of sensitivity for the measurement of turbidity is in therange of 15-20 μM β-AP. A decrease in turbidity over time in thepresence of the modulator, as compared to the turbidity in the absenceof the modulator, indicates that the modulator inhibits formation ofβ-AP fibers from monomeric β-AP. This assay can be performed usingstirring or shaking to accelerate polymerization, thereby increasing thespeed of the assay. Moreover the assay can be adapted to a 96-well plateformat to screen multiple compounds.

To perform the nucleation assay, first Aβ₁₋₄₀ peptide is dissolved inHFIP (1,1,1,3,3,3-Hexafluoro-2-propanol; Aldrich 10,522-8) at aconcentration of 2 mg peptide/ml and incubated at room temperature for30 min. HFIP-solubilized peptide is sonicated in a waterbath sonicatorfor 5 min at highest setting, then evaporated to dryness under a streamof argon. The peptide film is resuspended in anhydrous dimethylsulfoxide(DMSO) at a concentration of 6.9 mg/ml (25× concentration), sonicatedfor 5 min as before, then filtered through a 0.2 micron nylon syringefilter (VWR cat. No. 28196-050). Test compounds are dissolved in DMSO ata 100× concentration. Four volumes of 25×Aβ₁₋₄₀ peptide in DMSO arecombined with one volume of test compound in DMSO in a glass vial, andmixed to produce a 1:1 molar ratio of Aβ peptide to test compound. Fordifferent molar ratios, test compounds are diluted with DMSO prior toaddition to Aβ₁₋₄₀, in order to keep the final DMSO and Aβ₁₋₄₀concentrations constant. Control samples do not contain the testcompound. Ten microliters of the mixture is then added to the bottom ofa well of a Corning Costar ultra low binding 96-well plate (CorningCostar, Cambridge Mass.; cat. No. 2500). Ninety microliters of water isadded to the well, the plate is shaken on a rotary shaken at a constantspeed at room temperature for 30 seconds, an additional 100 μl of 2×PTLbuffer (20 mM NaH₂PO₄, 300 mM NaCl, pH 7.4) is added to the well, theplate is reshaken for 30 seconds and a baseline (t=0) turbidity readingis taken by measuring the apparent absorbance at 405 nm using a Bio-RadModel 450 Microplate Reader. The plate is then returned to the shakerand shaken continuously for 5 hours. Turbidity readings are taken at 15minute intervals.

β-amyloid aggregation in the absence of any modulators results inenhanced turbidity of the natural β-AP solution (i.e., an increase inthe apparent absorbance at 405 nm over time). Accordingly, a solutionincluding an effective inhibitory modulator compound exhibits reducedturbidity as compared to the control sample without the modulatorcompound (i.e., less apparent absorbance at 405 nm over time as comparedto the control sample).

B. Seeded Extension Assay

The seeded extension assay can be employed to measure the rate of Aβfiber formed in a solution of Aβ monomer following addition of polymericAβ fiber “seed”. The ability of test compounds to prevent furtherdeposition of monomeric Aβ to previously deposited amyloid is determinedusing a direct indicator of β-sheet formation using fluorescence. Incontrast with the nucleation assay, the addition of seed providesimmediate nucleation and continued growth of preformed fibrils withoutthe need for continuous mixing, and thus results in the absence of a lagtime before polymerization starts. Since this assay uses staticpolymerization conditions, the activity of positive compounds in thenucleation assay can be confirmed in this second assay under differentconditions and with an additional probe of amyloid structure.

In the seeded extension assay, monomeric Aβ₁₋₄₀ is incubated in thepresence of a “seed” nucleus (approximately ten mole percent of Aβ thathas been previously allowed to polymerize under controlled staticconditions). Samples of the solution are then diluted in thioflavin T(Th-T). The polymer-specific association of Th-T with Aβ produces afluorescent complex that allows the measurement of the extent of fibrilformation (Levine, H. (1993) Protein Science 2:404-410). In particular,association of Th-T with aggregated β-AP, but not monomeric or looselyassociated β-AP, gives rise to a new excitation (ex) maximum at 450 nmand an enhanced emission (em) at 482 nm, compared to the 385 nm (ex) and445 nm (em) for the free dye. Small aliquots of the polymerizationmixture contain sufficient fibril to be mixed with Th-T to allow themonitoring of the reaction mixture by repeated sampling. A linear growthcurve is observed in the presence of excess monomer. The formation ofthioflavin T responsive β-sheet fibrils parallels the increase inturbidity observed using the nucleation assay.

A solution of Aβ monomer for use in the seeded extension assay isprepared by dissolving an appropriate quantity of Aβ₁₋₄₀ peptide in 1/25volume of dimethylsulfoxide (DMSO), followed by water to ½ volume and ½volume 2× PBS (10× PBS: NaCl 137 mM, KCl 2.7 mM Na₂HPO₄.7H₂O 4.3 mM,KH₂PO₄ 1.4 mM pH 7.2) to a final concentration of 200 μM. To prepare thestock seed, 1 ml of the Aβ monomer preparation, is incubated forapproximately 8 days at 37° C. and sheared sequentially through an 18,23, 26 and 30 gauge needle 25, 25, 50, and 100 times respectively. 2 μlsamples of the sheared material is taken for fluorescence measurementsafter every 50 passes through the 30 gauge needle until the fluorescenceunits (FU) plateau (approx. 100-150×). Test compounds are prepared bydissolving an appropriate amount of test compound in 1× PBS to a finalconcentration of 1 mM (10× stock). If insoluble, the compound isdissolved in 1/10 volume of DMSO and diluted in 1× PBS to 1 mM. Afurther 1/10 dilution is also prepared to test each candidate at both100 μM and 10 μM.

To perform the seeded extension assay, each sample is set up with 50 μlof 200 μM monomer, 125 FU sheared seed (a variable quantity dependent onthe batch of seed, routinely 3-6 μl) and 10 μl of 10× modulatorsolution. The sample volume is then adjusted to a final volume of 100 μlwith 1×PBS. Two concentrations of each modulator typically are tested:100 μM and 10 μM, equivalent to a 1:1 and a 1:10 molar ratio of monomerto modulator. The controls include an unseeded reaction to confirm thatthe fresh monomer contains no seed, and a seeded reaction in the absenceof any modulators, as a reference to compare against candidatemodulators. The assay is incubated at 37° C. for 6 h, taking 2 μlsamples hourly for fluorescence measurements. To measure fluorescence, a2 μl sample of Aβ is added to 400 μl of Thioflavin-T solution (50 mMPotassium Phosphate 10 mM Thioflavin-T pH 7.5). The samples are vortexedand the fluorescence is read in a 0.5 ml micro quartz cuvette at EX 450nm and EM 482 nm (Hitachi 4500 Fluorimeter).

β-amyloid aggregation results in enhanced emission of Thioflavin-T.Accordingly, samples including an effective inhibitory modulatorcompound exhibit reduced emission as compared to control samples withoutthe modulator compound.

Example 7 Effect of Different Amino Acid Subregions of Aβ Peptide on theInhibitory Activity of β-Amyloid Modulator Compounds

To determine the effect of various subregions of Aβ₁₋₄₀ on theinhibitory activity of a a β-amyloid modulator, overlapping Aβ peptide15mers were constructed. For each 15mer, four different amino-terminalmodifiers were tested: a cholyl group, an iminobiotinyl group, anN-acetyl neuraminyl group (NANA) and a 5-(and 6-)-carboxyfluoresceinylgroup (FICO). The modulators were evaluated in the nucleation and seededextension assays described in Example 6.

The results of the nucleation assays are summarized below in Table II.The concentration of Aβ₁₋₄₀ used in the assays was 50 μM. The “mole %”value listed in Table II refers to the % concentration of the testcompound relative to Aβ₁₋₄₀. Accordingly, 100% indicates that Aβ₁₋₄₀ andthe test compound were equimolar. Mole % values less than 100% indicatethat Aβ₁₋₄₀ was in molar excess relative to the test compound (e.g., 10%indicates that Aβ₁₋₄₀ was in 10-fold molar excess relative to the testcompound). The results of the nucleation assays for each test compoundare presented in Table II in two ways. The “fold increase in lag time”,which is a measure of the ability of the compound to delay the onset ofaggregation, refers to the ratio of the observed lag time in thepresence of the test compound to the observed lag time in the controlwithout the test compound. Accordingly a fold increase in lag time of1.0 indicates no change in lag time, whereas numbers >1.0 indicate anincrease in lag time. The “% inhibition of plateau”, which is a measureof the ability of the compound to decrease the total amount ofaggregation, refers to the reduction of the final turbidity in thepresence of the test compound expressed as a percent of the controlwithout the test compound. Accordingly, an inhibitor that abolishesaggregation during the course of the assay will have a % inhibition of100. N-terminally modified Aβ subregions which exhibited inhibitoryactivity are indicated in bold in Table II.

TABLE II Fold % Reference N-terminal Mole Increase in Inhibition #Modification Aβ Peptide % Lag Time of Plateau PPI-174 cholyl Aβ ₁₋₁₅100 >4.5 100 PPI-264 cholyl Aβ ₆₋₂₀ 100 >4.5 100 PPI-269 cholyl Aβ₁₁₋₂₅100 1.5 ~0 PPI-274 cholyl Aβ ₁₆₋₃₀ 100 >4.5 100 PPI-279 cholyl Aβ ₂₁₋₃₅100 1.6 51 PPI-284 cholyl Aβ ₂₆₋₄₀ 100 >4.5 87 PPI-173 NANA Aβ₁₋₁₅ 100~1 ~0 PPI-266 NANA Aβ ₆₋₂₀ 100 1.3 64 PPI-271 NANA Aβ ₁₁₋₂₅ 100 1.3 77PPI-276 NANA Aβ₁₆₋₃₀ 100 ~1 ~0 PPI-281 NANA Aβ₂₁₋₃₅ 100 ~1 53 PPI-286NANA Aβ₂₆₋₄₀ 100 1.3 ~0 PPI-172 Iminobiotinyl Aβ₁₋₁₅ 100 1.2 ~0 PPI-267Iminobiotinyl Aβ ₆₋₂₀ 100 1.6 44 PPI-272 Iminobiotinyl Aβ ₁₁₋₂₅ 100 1.240 PPI-277 Iminobiotinyl Aβ ₁₆₋₃₀ 100 1.2 55 PPI-282 IminobiotinylAβ₂₁₋₃₅ 100 ~1 66 PPI-287 Iminobiotinyl Aβ₂₆₋₄₀ 100 2.3 ~0 PPI-190 FICOAβ₁₋₁₅ 100 ~1 30 PPI-268 FICO Aβ₆₋₂₀ 100 1.9 ~0 PPI-273 FICO Aβ ₁₁₋₂₅100 1.7 34 PPI-278 FICO Aβ ₁₆₋₃₀ 100 1.6 59 PPI-283 FICO Aβ ₂₁₋₃₅ 1001.2 25 PPI-288 FICO Aβ ₂₆₋₄₀ 100 2 75

These results indicate that certain subregions of Aβ₁₋₄₀, when modifiedwith an appropriate modifying group, are effective at inhibiting theaggregation of Aβ₁₋₄₀. A cholyl group was an effective modifying groupfor several subregions. Cholic acid alone was tested for inhibitoryactivity but had no effect on Aβ aggregation. The Aβ₆₋₂₀ subregionexhibited high levels of inhibitory activity when modified with severaldifferent modifying groups (cholyl, NANA, iminobiotinyl), withcholyl-Aβ₆₋₂₀ (PPI-264) being the most active form. Accordingly, thismodulator compound was chosen for further analysis, described in Example8.

Example 8 Identification of a Five Amino Acid Subregion of Aβ PeptideSufficient for Inhibitory Activity of a β-Amyloid Modulator Compound

To further delineate a minimal subregion of cholyl-Aβ₆₋₂₀ sufficient forinhibitory activity, a series of amino terminal and carboxy terminalamino acid deletions of cholyl-Aβ₆₋₂₀ were constructed. The modulatorsall had the same cholyl amino-terminal modification. Additionally, forthe peptide series having carboxy terminal deletions, the carboxyterminus was further modified to an amide. The modulators were evaluatedas described in Example 7 and the results are summarized below in TableIII, wherein the data is presented as described in Example 7.

TABLE III Fold % N-Term. Aβ C-Term. Mole Increase in Inhibition Ref. #Mod. Peptide Mod. % Lag Time of Plateau PPI-264 cholyl Aβ ₆₋₂₀ —100 >4.5 100 10 2 43 PPI-341 cholyl Aβ ₇₋₂₀ — 100 >4.5 100 33 2 ~0PPI-342 cholyl Aβ₈₋₂₀ — 100 1.5 ~0 33 2.1 ~0 PPI-343 cholyl Aβ₉₋₂₀ — 332.0 ~0 PPI-344 cholyl Aβ₁₀₋₂₀ — 33 2.1 ~0 PPI-345 cholyl Aβ₁₁₋₂₀ — 331.5 ~0 PPI-346 cholyl Aβ₁₂₋₂₀ — 33 2.1 ~0 PPI-347 cholyl Aβ₁₃₋₂₀ — 332.6 ~0 PPI-348 cholyl Aβ ₁₄₋₂₀ — 33 2.0 49 PPI-349 cholyl Aβ ₁₅₋₂₀ — 332.3 50 PPI-350 cholyl Aβ ₁₆₋₂₀ — 38 3.4 23 PPI-296 cholyl Aβ₆₋₂₀ amide33 1.8 ~0 PPI-321 cholyl Aβ₆₋₁₉ amide 33 1.4 ~0 PPI-325 cholyl Aβ₆₋₁₇amide 33 1.8 ~0 PPI-331 cholyl Aβ₆₋₁₄ amide 33 1.0 29 PPI-339 cholylAβ₆₋₁₀ amide 33 1.1 13

These results indicate that activity of the modulator is maintained whenamino acid residue 6 is removed from the amino terminal end of themodulator (i.e., cholyl-Aβ₇₋₂₀ retained activity) but activity is lostas the peptide is deleted further at the amino-terminal end by removalof amino acid position 7 through to amino acid position 12 (i.e.,cholyl-Aβ₈₋₂₀ through cholyl-Aβ₁₃₋₂₀ did inhibit the plateau level of Aβaggregation). However, further deletion of amino acid position 13resulted in a compound (i.e., cholyl-Aβ₁₄₋₂₀) in which inhibitoryactivity is restored. Furthermore, additional deletion of amino acidposition 14 (i.e., cholyl-Aβ₁₅₋₂₀) or positions 14 and 15 (i.e.,cholyl-Aβ₁₆₋₂₀) still maintained inhibitory activity. Thus, aminoterminal deletions of Aβ₆₋₂₀ identified Aβ₁₆₋₂₀ as a minimal subregionwhich is sufficient for inhibitory activity when appropriately modified.In contrast, carboxy terminal deletion of amino acid position 20resulted in loss of activity which was not fully restored as the peptidewas deleted further at the carboxy-terminal end. Thus, maintenance ofposition 20 within the modulator may be important for inhibitoryactivity.

Example 9 Identification of a Four Amino Acid Subregion of Aβ PeptideSufficient for Inhibitory Activity of a β-Amyloid Modulator Compound

In this example, the smallest effective modulator identified in thestudies described in Example 8, cholyl-Aβ₁₆₋₂₀ (PPI-350), was analyzedfurther. Additional amino- and carboxy-terminal deletions were made withcholyl-Aβ₁₆₋₂₀, as well as an amino acid substitution (Val₁₈→Thr), toidentify the smallest region sufficient for the inhibitory activity ofthe modulator. A peptide comprised of five alanine residues, (Ala)₅, SEQID NO: 35, modified at its amino-terminus with cholic acid, was used asa specificity control. The modulators were evaluated as described inExample 7 and the results are summarized below in Table IV, wherein thedata is presented as described in Example 7.

TABLE IV C- Fold % N-Term. Aβ Term. Mole Increase in Inhibition Ref. #Mod. Peptide Mod. % Lag Time of Plateau PPI-264 cholyl Aβ ₆₋₂₀ — 10 2.043 PPI-347 cholyl Aβ ₁₃₋₂₀ — 10 2.2 57 PPI-349 cholyl Aβ ₁₅₋₂₀ —100 >5.0 100 33 2.6 35 10 2.1 ~0 PPI-350 cholyl Aβ ₁₆₋₂₀ — 100 >5.0 10010 2.4 40 PPI-368 cholyl Aβ ₁₇₋₂₁ — 100 >5.0 100 PPI-374 imino- Aβ ₁₆₋₂₀— 100 1.3 86 biotinyl PPI-366 cholyl Aβ₁₅₋₁₉ — 100 3.1 ~0 10 1.6 ~0PPI-369 cholyl Aβ₁₆₋₂₀ — 100 ~1 ~0 (Val₁₈−> Thr) PPI-370 cholyl Aβ ₁₆₋₂₀— 100 2.6 73 (Phe ₁₉ −> Ala) PPI-365 cholyl (Ala)₅ — 100 ~1 ~0 PPI-319cholyl Aβ ₁₆₋₂₀ amide 33 5.6 ~0 10 2.7 ~0 PPI-321 cholyl Aβ₁₆₋₁₉ amide100 1.2 ~0 PPI-377 — Aβ₁₆₋₂₀ — 100 ~1 ~0

As shown in Table IV, cholyl-Aβ₁₆₋₂₀ (PPI-350) and cholyl-Aβ₁₇₋₂₁(PPI-368) both exhibited inhibitory activity, indicating that thefour-amino acid minimal subregion of positions 17-20 is sufficient forinhibitory activity. Loss of position 20 (e.g., in PPI-366 and PPI-321)resulted in loss of inhibitory activity, demonstrating the importance ofposition 20. Moreover, mutation of valine at position 18 to threonine(in PPI-369) also resulted in loss of activity, demonstrating theimportance of position 18. In contrast, mutation of phenylalanine atposition 19 to alanine (cholyl-Aβ₁₆₋₂₀ Phe₁₉→Ala; PPI-370) resulted in acompound which still retained detectable inhibitory activity.Accordingly, the phenylalanine at position 19 is more amenable tosubstitution, preferably with another hydrophobic amino acid residue.Cholyl-penta-alanine (SEQ ID NO: 35) (PPI-365) showed no inhibitoryactivity, demonstrating the specificity of the Aβ peptide portion of themodulator. Moreover, unmodified Aβ₁₆₋₂₀ (PPI-377) was not inhibitory,demonstrating the functional importance of the amino-terminal modifyinggroup. The specific functional group influenced the activity of themodulator. For example, iminobiotinyl-Aβ₁₆₋₂₀ (PPI-374) exhibitedinhibitory activity similar to cholyl-Aβ₁₆₋₂₀, whereas an N-acetylneuraminic acid (NANA)-modified Aβ₁₆₋₂₀ was not an effective inhibitorymodulator (not listed in Table IV). A C-terminal amide derivative ofcholyl-Aβ₁₆₋₂₀ (PPI-319) retained high activity in delaying the lag timeof aggregation, indicating that the carboxy-terminus of the modulatorcan be derivatized without loss of inhibitory activity. Although thisamide-derivatized compound did not inhibit the overall plateau level ofaggregation over time, the compound was not tested at concentrationshigher than mole 33%. Higher concentrations of the amide-derivatizedcompound are predicted to inhibit the overall plateau level ofaggregation, similar to cholyl-Aβ₁₆₋₂₀ (PPI-350).

Example 10 Effect of β-Amyloid Modulators on the Neurotoxicity ofNatural β-Amyloid Peptide Aggregates

The neurotoxicity of natural β-amyloid peptide aggregates, in either thepresence or absence of a β-amyloid modulator, is tested in a cell-basedassay using either a rat or human neuronally-derived cell line (PC-12cells or NT-2 cells, respectively) and the viability indicator3,(4,4-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT). (Seee.g., Shearman, M. S. et al (1994) Proc. Natl. Acad. Sci. USA91:1470-1474; Hansen, M. B. et al. (1989) J. Immun. Methods 119:203-210for a description of similar cell-based viability assays). PC-12 is arat adrenal pheochromocytoma cell line and is available from theAmerican Type Culture Collection, Rockville, Md. (ATCC CRL 1721). MTT(commercially available from Sigma Chemical Co.) is a chromogenicsubstrate that is converted from yellow to blue in viable cells, whichcan be detected spectrophotometrically.

To test the neurotoxicity of natural β-amyloid peptides, stock solutionsof fresh Aβ monomers and aged Aβ aggregates were first prepared. Aβ₁₋₄₀in 100% DMSO was prepared from lyophilized powder and immediatelydiluted in one half the final volume in H₂O and then one half the finalvolume in 2×PBS so that a final concentration of 200 μM peptide, 4% DMSOis achieved. Peptide prepared in this way and tested immediately oncells is referred to as “fresh” Aβ monomer. To prepare “aged” Aβaggregates, peptide solution was placed in a 1.5 ml Eppendorf tube andincubated at 37° C. for eight days to allow fibrils to form. Such “aged”Aβ peptide can be tested directly on cells or frozen at −80° C. Theneurotoxicity of fresh monomers and aged aggregates were tested usingPC12 and NT2 cells. PC12 cells were routinely cultured in Dulbeco'smodified Eagle's medium (DMEM) containing 10% horse serum, 5% fetal calfserum, 4 mM glutamine, and 1% gentamycin. NT2 cells were routinelycultured in OPTI-MEM medium (GIBCO BRL CAT. #31985) supplemented with10% fetal calf serum, 2 mM glutamine and 1% gentamycin. Cells wereplated at 10-15,000 cells per well in 90 μl of fresh medium in a 96-welltissue culture plate 3-4 hours prior to treatment. The fresh or aged Aβpeptide solutions (10 μL) were then diluted 1:10 directly into tissueculture medium so that the final concentration was in the range of 1-10μM peptide. Cells are incubated in the presence of peptide without achange in media for 48 hours at 37° C. For the final three hours ofexposure of the cells to the β-AP preparation, MTT was added to themedia to a final concentration of 1 mg/ml and incubation was continuedat 37° C. Following the two hour incubation with MTT, the media wasremoved and the cells were lysed in 100 μL isopropanol/0.4N HCl withagitation. An equal volume of PBS was added to each well and the plateswere agitated for an additional 10 minutes. Absorbance of each well at570 nm was measured using a microtiter plate reader to quantitate viablecells.

The neurotoxicity of aged (5 day or 8 day) Aβ₁₋₄₀ aggregates alone, butnot fresh Aβ₁₋₄₀ monomers alone, was confirmed in an experiment theresults of which are shown in FIG. 3, which demonstrates that incubatingthe neuronal cells with increasing amounts of fresh Aβ₁₋₄₀ monomers wasnot significantly toxic to the cells whereas incubating the cells withincreasing amounts of 5 day or 8 day Aβ₁₋₄₀ aggregates led to increasingamount of neurotoxicity. The EC50 for toxicity of aged Aβ₁₋₄₀ aggregateswas 1-2 μM for both the PC12 cells and the NT2 cells.

To determine the effect of a β-amyloid modulator compound on theneurotoxicity of Aβ₁₋₄₀ aggregates, a modulator compound, cholyl-Aβ₆₋₂₀(PPI-264), was preincubated with Aβ₁₋₄₀ monomers under standardnucleation assay conditions as described in Example 6 and at particulartime intervals post-incubation, aliquots of the β-AP/modulator solutionwere removed and 1) the turbidity of the solution was assessed as ameasure of aggregation and 2) the solution was applied to culturedneuronal cells for 48 hours at which time cell viability was assessedusing MTT to determine the neurotoxicity of the solution. The results ofthe turbidity analysis are shown in FIG. 4, panels A, B and C. In panelA, Aβ₁₋₄₀ and cholyl-Aβ₆₋₂₀ were both present at 64 μM. In panel B,Aβ₁₋₄₀ was present at 30 μM and cholyl-Aβ₆₋₂₀ was present at 64 μM. Inpanel C, Aβ₁₋₄₀ was present at 10 μM and cholyl-Aβ₆₋₂₀ was present at 64μM. These data show that an equimolar amount of cholyl-Aβ₆₋₂₀ iseffective at inhibiting aggregation of Aβ₁₋₄₀ (see FIG. 4, panel A) andthat as the concentration of Aβ₁₋₄₀ is reduced, the amount of detectableaggregation of the Aβ₁₋₄₀ monomer is correspondingly reduced (compareFIG. 4, panels B and C with panel A). The corresponding results of theneurotoxicity analysis are shown in FIG. 4, panels D, E, and F. Theseresults demonstrate that the β-amyloid modulator compound not onlyinhibits aggregation of Aβ₁₋₄₀ monomers but also inhibits theneurotoxicity of the Aβ₁₋₄₀ solution, illustrated by the reduced percenttoxicity of the cells when incubated with the Aβ₁₋₄₀/modulator solutionas compared to Aβ₁₋₄₀ alone (see e.g., FIG. 4, panel D). Moreover, evenwhen Aβ₁₋₄₀ aggregation was not detectable as measured by lightscattering, the modulator compound inhibited the neurotoxicity of theAβ₁₋₄₀ solution (see. FIG. 4, panels E and F). Thus, the formation ofneurotoxic Aβ₁₋₄₀ aggregates precedes the formation of insolubleaggregates detectable by light scattering and the modulator compound iseffective at inhibiting the inhibiting the formation and/or activity ofthese neurotoxic aggregates. Similar results were seen with othermodulator compounds, such as iminobiotinyl-Aβ₆₋₂₀ (PPI-267),cholyl-Aβ₁₆₋₂₀ (PPI-350) and cholyl-Aβ₁₆₋₂₀-amide (PPI-319).

Additionally, the β-amyloid modulator compounds have been demonstratedto reduce the neurotoxicity of preformed Aβ₁₋₄₀ aggregates. In theseexperiments, Aβ₁₋₄₀ aggregates were preformed by incubation of themonomers in the absence of any modulators. The modulator compound wasthen incubated with the preformed Aβ₁₋₄₀ aggregates for 24 hours at 37°C., after which time the β-AP/modulator solution was collected and itsneurotoxicity evaluated as described above. Incubation of preformedAβ₁₋₄₀ aggregates with the modulator compound prior to applying thesolution to neuronal cells resulted in a decrease in the neurotoxicityof the Aβ₁₋₄₀ solution. These results suggest that the modulator caneither bind to Aβ fibrils or soluble aggregate and modulate theirinherent neurotoxicity or that the modulator can perturb the equilibriumbetween monomeric and aggregated forms of Aβ₁₋₄₀ in favor of thenon-neurotoxic form.

Example 11 Characterization of Additional β-Amyloid Modulator Compounds

In this example, additional modulator compounds designed based uponamino acids 17-20 of Aβ, LVFF (SEQ ID NO: 12) (identified in Example 9),were prepared and analyzed to further delineate the structural featuresnecessary for inhibition of β-amyloid aggregation. Types of compoundsanalyzed included ones having only three amino acid residues of anaggregation core domain, compounds in which the amino acid residues ofan Aβ aggregation core domain were rearranged or in which amino acidsubstitutions had been made, compounds modified with a carboxy-terminalmodifying group and compounds in which the modifying group had beenderivatized. Abbreviations used in this example are: h-(free aminoterminus), -oh (free carboxylic acid terminus), -nh₂ (amide terminus),CA (cholyl, the acyl portion of cholic acid), NANA (N-acetylneuraminyl), IB (iminobiotinyl), βA (β-alanyl), DA (D-alanyl), Adp(aminoethyldibenzofuranylpropanoic acid), Aic(3-(O-aminoethyl-iso)-cholyl, a derivative of cholic acid), IY(iodotyrosyl), o-methyl (carboxy-terminal methyl ester), N-me (N-methylpeptide bond), DeoxyCA (deoxycholyl) and LithoCA (lithocholyl).

Modulator compounds having an Aic modifying group at either the amino-or carboxy-terminus (e.g., PPI-408 and PPI-418) were synthesized usingknown methods (see e.g., Wess, G. et al. (1993) Tetrahedron Letters,34:817-822; Wess, G. et al. (1992) Tetrahedron Letters 33:195-198).Briefly, 3-iso-O-(2-aminoethyl)-cholic acid(3β-(2-aminoethoxy)-7α,12α-dihydroxy-5β-cholanoic acid) was converted tothe FMOC-protected derivative using FMOC-OSu (the hydroxysuccinimideester of the FMOC group, which is commercially available) to obtain areagent that was used to introduce the cholic acid derivative into thecompound. For N-terminal introduction of the cholic acid moiety, theFMOC-protected reagent was coupled to the N-terminal amino acid of asolid-phase peptide in the standard manner, followed by standardFMOC-deprotection conditions and subsequent cleavage from the resin,followed by HPLC purification. For C-terminal introduction of the cholicacid moiety, the FMOC-protected reagent was attached to 2-chlorotritylchloride resin in the standard manner. This amino acyl derivatized resinwas then used in the standard manner to synthesize the complete modifiedpeptide.

The modulators were evaluated in the nucleation and seeded extensionassays described in Example 6 and the results are summarized below inTable V. The change in lag time (ΔLag) is presented as the ratio of thelag time observed in the presence of the test compound to the lag timeof the control. Data are reported for assays in the presence of 100 mole% inhibitor relative to the concentration of Aβ₁₋₄₀, except for PPI-315,PPI-348, PPI-380, PPI-407 and PPI-418, for which the data is reported inthe presence of 33 mole % inhibitor. Inhibition (% I_(nucl'n)) is listedas the percent reduction in the maximum observed turbidity in thecontrol at the end of the assay time period. Inhibition in the extensionassay (% I_(ext'n)) is listed as the percent reduction of thioflavin-Tfluorescence of β-structure in the presence of 25 mole % inhibitor.Compounds with a % I_(nucl'n) of at least 30% are highlighted in bold.

TABLE V N-Term. C-Term. % % Ref. # Mod. Peptide Mod. ΔLag I_(nucl'n)I_(ext'n) PPI-293 CA — -oh 1.0    0  ND* PPI-315 CA HQKLVFF -nh₂ 1.1   5** ND (SEQ ID NO: 6) PPI-316 NANA HQKLVFF -nh₂ 1.5  −15 ND (SEQ IDNO: 6) PPI-319 CA KLVFF -nh ₂ 5.4    70 52 (SEQ ID NO: 10) PPI-339 CAHDSGY -nh₂ 1.1  −18 ND (SEQ ID NO: 36) PPI-348 CA HQKLVFF -oh 2.0   70** ND (SEQ ID NO: 6) PPI-349 CA QKLVFF -oh >5   100 56 (SEQ ID NO:8) PPI-350 CA KLVFF -oh 1.8    72 11 (SEQ ID NO: 10) PPI-365 CA AAAAA-oh 0.8  −7 0 (SEQ ID NO: 35) PPI-366 CA QKLVF -oh 3.1  −23 ND (SEQ IDNO: 6) PPI-368 CA LVFFA -oh >5   100 91 (SEQ ID NO: 11) PPI-369 CA KLTFFOh 1.1  −16 44 (SEQ ID NO: 38) PPI-370 CA KLVAF -oh 2.6    73 31 (SEQ IDNO: 30) PPI-371 CA KLVFF(βA) -oh 2.5    76 80 (SEQ ID NO: 31) PPI-372 CAFKFVL -oh 0.8    45 37 (SEQ ID NO: 29) PPI-373 NANA KLVFF -oh 0.9    168 (SEQ ID NO: 10) PPI-374 IB KLVFF -oh 1.3    86 0 (SEQ ID NO: 10)PPI-375 CA KTVFF -oh 1.2    18 21 (SEQ ID NO: 39) PPI-377 h- KLVFF -oh1.1    0 8 (SEQ ID NO: 10) PPI-379 CA LVFFAE -oh 1.4    55 16 (SEQ IDNO: 27) PPI-380 CA LVFF -oh 1.8    72** 51 (SEQ ID NO: 12) PPI-381 CALVFF(DA) -oh 2.3    56 11 (SEQ ID NO: 6) PPI-382 CA LVFFA -nh₂ 1.0 −20091 (SEQ ID NO: 11) PPI-383 h-DDIIL- VFF -oh 0.4    14 0 (Adp) PPI-386 h-LVFFA -oh 1.0    15 11 (SEQ ID NO: 11) PPI-387 h- KLVFF -nh₂ 1.3  −9 39(SEQ ID NO: 10) PPI-388 CA AVFFA -oh 1.4    68 44 (SEQ ID NO: 25)PPI-389 CA LAFFA -oh 1.5    47 66 (SEQ ID NO: 13) PPI-390 CA LVAFA -oh2.7    25 0 (SEQ ID NO: 33) PPI-392 CA VFFA -oh 2.0    76 10 (SEQ ID NO:24) PPI-393 CA LVF -oh 1.3    1 0 PPI-394 CA VFF -oh 1.8    55 0 PPI-395CA FFA -oh 1.0    51 6 PPI-396 CA LV(IY)FA -oh >5   100 71 (SEQ ID NO:23) PPI-401 CA LVFFA -o-methyl ND ND 0 (SEQ ID NO: 11) PPI-405 h- LVFFA-nh₂ 1.3    11 70 (SEQ ID NO: 11) PPI-407 CA LVFFK -oh >5   100** 85(SEQ ID NO: 22) PPI-408 h- LVFFA (Aic)-oh 3.5    46 3 (SEQ ID NO: 11)PPI-418 h-(Aic) LVFFA -oh >5   100** 87 (SEQ ID NO: 11) PPI-426 CA FFVLA-oh >5   100 89 (SEQ ID NO: 40) PPI-391 CA LVFAA -oh 1.6    40 ND (SEQID NO: 41) PPI-397 CA LVF(IY)A -oh >5    95 ND (SEQ ID NO: 26) PPI-400CA AVAFA -oh 1.0  −15 ND (SEQ ID NO: 42) PPI-403 *** HQKLVFF -oh 1.4 −75 0 (SEQ ID NO: 6) PPI-404 **** LKLVFF -oh 1.8  −29 7 (SEQ ID NO: 43)PPI-424 DeoxyCA LVFFA -oh 3.0 −114 82 (SEQ ID NO: 11) PPI-425 LithoCALVFFA -oh 2.8 −229 0 (SEQ ID NO: 11) PPI-428 CA FF -oh 1.7  −78 15PPI-429 CA FFV -oh 2.2  −33 7 PPI-430 CA FFVL -oh 4.1    33 75 (SEQ IDNO: 28) PPI-433 CA LVFFA -oh 2.8    27 ND (all D amino acids) PPI-435t-Boc LVFFA -oh 3.0  −5 ND (SEQ ID NO: 11) PPI-438 CA GFF -oh 1.0    0ND *ND = not done **= 33 mol % ***= h-DDIII(N-Me-Val)DLL(Adp) (SEQ IDNO: 44) ****= h-DDII(N-Me-Leu)VEH(Adp) (SEQ ID NO: 45)

Certain compounds shown in Table V (PPI-319, PPI-349, PPI-350, PPI-368and PPI-426) also were tested in neurotoxicity assays such as thosedescribed in Example 10. For each compound, the delay of the appearanceof neurotoxicity relative to control coincided with the delay in thetime at which polymerization of Aβ began in the nucleation assays. Thiscorrelation between the prevention of formation of neurotoxic Aβ speciesand the prevention of polymerization of Aβ was consistently observed forall compounds tested.

The results shown in Table V demonstrate that at an effective modulatorcompound can comprise as few as three Aβ amino acids residues (seePPI-394, comprising the amino acid sequence VFF, which corresponds toAβ₁₈₋₂₀, and PPI-395, comprising the amino acid sequence FFA, whichcorresponds to Aβ₁₉₋₂₁). The results also demonstrate that a modulatorcompound having a modulating group at its carboxy-terminus is effectiveat inhibiting Aβ aggregation (see PPI-408, modified at its C-terminuswith Aic). Still further, the results demonstrate that the cholyl group,as a modulating group, can be manipulated while maintaining theinhibitory activity of the compounds (see PPI-408 and PPI-418, both ofwhich comprise the cholyl derivative Aic). The free amino group of theAic derivative of cholic acid represents a position at which a chelationgroup for ^(99m)Tc can be introduced, e.g., to create a diagnosticagent. Additionally, the ability to substitute iodotyrosyl forphenylalanine at position 19 or 20 of the Aβ sequence (see PPI-396 andPPI-397) while maintaining the ability of the compound to inhibit Aβaggregation indicates that the compound could be labeled withradioactive iodine, e.g., to create a diagnostic agent, without loss ofthe inhibitory activity of the compound.

Finally, compounds with inhibitory activity were created using Aβderived amino acids but wherein the amino acid sequence was rearrangedor had a substitution with a non-Aβ-derived amino acid. Examples of suchcompounds include PPI-426, in which the sequence of Aβ₁₇₋₂₁ (LVFFA SEQID NO: 11) has been rearranged (FFVLA SEQ ID NO: 40), PPI-372, in whichthe sequence of Aβ₁₆₋₂₀ (KLVFF SEQ ID NO: 10) has been rearranged (FKFVLSEQ ID NO: 29), and PPI-388, -389 and -390, in which the sequence ofAβ₁₇₋₂₁ (LVFFA SEQ ID NO: 11) has been substituted at position 17, 18 or19, respectively, with an alanine residue (AVFFA (SEQ ID NO: 25) forPPI-388, LAFFA (SEQ ID NO: 13) for PPI-389 and LVAFA (SEQ ID NO: 33) forPPI-390). The inhibitory activity of these compounds indicate that thepresence in the compound of an amino acid sequence directlycorresponding to a portion of Aβ is not essential for inhibitoryactivity, but rather suggests that maintenance of the hydrophobic natureof this core region, by inclusion of amino acid residues such asphenylalanine, valine, leucine, regardless of their precise order, canbe sufficient for inhibition of Aβ aggregation.

Example 12 Characterization of β-Amyloid Modulator Compounds Comprisingan Unmodified β-Amyloid Peptide

To examine the ability of unmodified Aβ peptides to modulate aggregationof natural β-AP, a series of Aβ peptides having amino- and/or carboxyterminal deletions as compared to Aβ₁₋₄₀, or having internal amino acidsdeleted (i.e., noncontiguous peptides), were prepared. One peptide(PPI-220) had additional, non-Aβ-derived amino acid residues at itsamino-terminus. These peptides all had a free amino group at theamino-terminus and a free carboxylic acid at the carboxy-terminus. Theseunmodified peptides were evaluated in assays as described in Example 7.The results are summarized below in Table VI, wherein the data ispresented as described in Example 7. Compounds exhibiting at least a 1.5fold increase in lag time are highlighted in bold.

TABLE VI Fold Reference Mole Increase in % Inhibition # Aβ Peptide % LagTime of Plateau PPI-226 Aβ ₆₋₂₀ 100 1.66 76 PPI-227 Aβ₁₁₋₂₅ 100 ~1 47PPI-228 Aβ ₁₆₋₃₀ 100 >4.5 100 PPI-229 Aβ₂₁₋₃₅ 100 ~1 ~0 PPI-230 Aβ₂₆₋₄₀100 0.8 ~0 PPI-231 Aβ₁₋₁₅ 100 ~1 18 PPI-247 Aβ_(1-30, 36-40)(Δ31-35) 100~1 ~0 PPI-248 Aβ _(1-25, 31-40) (Δ26-30) 100 1.58 ~0 PPI-249 Aβ_(1-20, 26-40) (Δ21-25) 100 2.37 ~0 PPI-250 Aβ _(1-15, 21-40) (Δ16-20)100 1.55 ~0 PPI-251 Aβ_(1-10, 16-40)(Δ11-15) 100 ~1.2 ~0 PPI-252 Aβ_(1-5, 11-40) (Δ6-10) 100 1.9 33 PPI-253 Aβ ₆₋₄₀ 100 1.9 ~0 PPI-220EEVVHHHHQQ-Aβ ₁₆₋₄₀ 100 >4 100

The results shown in Table VI demonstrate that limited portions of theAβ sequence can have a significant inhibitory effect on natural β-APaggregation even when the peptide is not modified by a modifying group.Preferred unmodified peptides are Aβ₆₋₂₀ (PPI-226), Aβ₁₆₋₃₀ (PPI-228),Aβ_(1-20, 26-40) (PPI-249) and EEVVHHHHQQ-Aβ₁₆₋₄₀ (SEQ ID NO: 16)(PPI-220), the amino acid sequences of which are shown in SEQ ID NOs: 4,14, 15, and 16, respectively.

Forming part of this disclosure is the appended Sequence Listing, thecontents of which are summarize in the Table below.

SEQ ID NO: Amino Acids Peptide Sequence 1 43 amino acids Aβ₁₋₄₃ 2 103amino acids APP C-terminus 3 43 amino acids Aβ₁₋₄₃ (19, 20 mutated) 4HDSGYEVHHQKLVFF Aβ₆₋₂₀ 5 HQKLVFFA Aβ₁₄₋₂₁ 6 HQKLVFF Aβ₁₄₋₂₀ 7 QKLVFFAAβ₁₅₋₂₁ 8 QKLVFF Aβ₁₅₋₂₀ 9 KLVFFA Aβ₁₆₋₂₁ 10 KLVFF Aβ₁₆₋₂₀ 11 LVFFAAβ₁₇₋₂₁ 12 LVFF Aβ₁₇₋₂₀ 13 LAFFA Aβ₁₇₋₂₁ (V₁₈→A) 14 KLVFFAEDVGSNKGAAβ₁₆₋₃₀ 15 35 amino acids Aβ_(1-20, 26-40) 16 35 amino acidsEEVVHHHHQQ-βAP₁₆₋₄₀ 17 AGAAAAGA PrP peptide 18 AILSS amylin peptide 19VFF Aβ₁₈₋₂₀ 20 FFA Aβ₁₉₋₂₁ 21 FFVLA Aβ₁₇₋₂₁ (scrambled) 22 LVFFK Aβ₁₇₋₂₁(A₂₁→K) 23 LV(IY)FA Aβ₁₇₋₂₁ (F₁₉→IY) 24 VFFA Aβ₁₈₋₂₁ 25 AVFFA Aβ₁₇₋₂₁(L₁₇→A) 26 LVF(IY)A Aβ₁₇₋₂₁ (F₂₀→IY) 27 LVFFAE Aβ₁₇₋₂₂ 28 FFVL Aβ₁₇₋₂₀(scrambled) 29 FKFVL Aβ₁₆₋₂₀ (scrambled) 30 KLVAF Aβ₁₆₋₂₀ (F₁₉→A) 31KLVFF(βA) Aβ₁₆₋₂₁ (A₂₁→βA) 32 LVFF(DA) Aβ₁₇₋₂₁ (A₂₁→DA) 33 LVAFA 34DDIIL-Adp 35 AAAAA 36 HDSGY 37 QKLVF 38 KLTFF 39 KTVFF 40 FFVLA 41 LVFAA42 AVAFA 43 LKLVFF 44 DDIII-(N-Me-Val)-DLL-(Adp) 45DDII-(N-Me-Leu)-VEH-(Adp)

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An amyloid modulator compound having the structure:

wherein Xaa is an amyloidogenic protein, or peptide fragment thereof, ofat least 4 amino acid residues in length and A is a modifying groupcomprising a cyclic, heterocyclic or polycyclic group, covalentlyattached to the carboxy-terminus of the amyloidogenic protein, orpeptide fragment thereof, such that the compound modulates theaggregation of natural amyloid proteins or peptides when contacted withthe natural amyloidogenic proteins or peptides, wherein Xaa is notcalcitonin.
 2. An amyloid modulator compound comprising the structure:

wherein Xaa is an amyloidogenic protein, or peptide fragment thereof, ofat least 4 amino acid residues in length and A is a modifying groupcomprising a cyclic, heterocyclic or polycyclic group, covalentlyattached to the α-amino group at the amino-terminus of the amyloidogenicprotein, or peptide fragment thereof, such that the compound modulatesthe aggregation of natural amyloid proteins or peptides when contactedwith the natural amyloidogenic proteins or peptides, wherein Xaa isselected from the group consisting of transthyretin (TTR), prion protein(PrP), atrial natriuretic factor (ANF), kappa light chain, lambda lightchain, amyloid A, procalcitonin, cystatin C, β2 microglobulin, ApoA-J,gelsolin, fibrinogen and lysozyme.
 3. The compound of claim 2, whichinhibits aggregation of natural amyloidogenic proteins or peptides whencontacted with the natural amyloidogenic proteins or peptides.
 4. Thecompound of claim 3, which inhibits aggregation of natural amyloidogenicproteins or peptides when contacted with a molar excess amount ofnatural amyloidogenic proteins or peptides.
 5. The compound of claim 1or 2, wherein the modifying group contains a cis-decalin group.
 6. Thecompound of claim 5, wherein the modifying group contains a cholanoylstructure.
 7. The compound of claim 6, wherein the modifying group is acholyl group.
 8. The compound of claim 1 or 2, wherein the modifyinggroup comprises a biotin-containing group, adiethylene-triaminepentaacetyl group, a (−)-menthoxyacetyl group, afluorescein-containing group or an N-acetylneuraminyl group.
 9. Thecompound of claim 1, which inhibits aggregation of natural amyloidogenicproteins or peptides when contacted with the natural amyloidogenicproteins or peptides.
 10. The compound of claim 1, which is furthermodified to alter a pharmacokinetic property of the compound.
 11. Thecompound of claim 1, which is further modified to label the compoundwith a detectable substance.
 12. The compound of claim 1, wherein theamyloidogenic protein, or peptide fragment thereof, is selected from thegroup consisting of transthyretin (TTR), prion protein (PrP), isletamyloid polypeptide (IAPP), atrial natriuretic factor (ANF), kappa lightchain, lambda light chain, amyloid A, procalcitonin, cystatin C, β2microglobulin, ApoA-J, gelsolin, fibrinogen and lysozyme.
 13. Apharmaceutical composition comprising a therapeutically effective amountof the compound of claim 1 or 2 and a pharmaceutically acceptablecarrier.
 14. A method for altering aggregation of natural amyloidproteins or peptides, comprising contacting the natural amyloid proteinsor peptides with the compound of claim 1 or 2 such that aggregation ofthe natural amyloid proteins or peptides is altered.
 15. A method fordetecting aggregation of natural amyloid proteins or peptides,comprising: (1) modifying the compound of claim 1 or 2 with a detectablesubstance; and (2) contacting a biological sample with the modifiedcompound such that aggregation of the natural amyloid proteins orpeptides in the sample is detected.
 16. The method of claim 15, whereinthe compound is administered to a subject to detect aggregation of thenatural amyloid proteins or peptides in the subject.
 17. The method ofclaim 16, wherein the compound is labeled with radioactive iodine ortechnetium.
 18. A method for treating a subject for a disorderassociated with amyloidosis, comprising: administering to the subject atherapeutically or prophylactically effective amount of the compound ofclaim 2 such that the subject is treated for a disorder associated withamyloidosis.
 19. A method for treating a subject for a disorderassociated with amyloidosis, comprising: administering to the subject atherapeutically or prophylactically effective amount of the compound ofclaim 1 such that the subject is treated for a disorder associated withamyloidosis.
 20. The method of claim 19, wherein the disorder isselected from the group consisting of familial amyloid polyneuropathy,familial amyloid cardiomyopathy, isolated cardiac amyloid, systemicsenile amyloidosis, scrapie, bovine spongiform encephalopathy,Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome,adult onset diabetes, insulinoma, isolated atrial amyloidosis,idiopathic amyloidosis, myeloma or macroglobulinemia-associatedamyloidosis, primary localized cutaneous nodular amyloidosis associatedwith Sjogren's syndrome, reactive amyloidosis, familial MediterraneanFever and familial amyloid nephropathy with urticaria and deafness,hereditary cerebral hemorrage with amyloidosis of Icelandic type,amyloidosis associated with long term hemodialysis, hereditarynon-neuropathic systemic amyloidosis, familial amyloidosis of Finnishtype, amyloidosis associated with medullary carcinoma of the thyroid,fibrinogen-associated hereditary renal amyloidosis andlysozyme-associated hereditary systemic amyloidosis.